Using synchronized tobacco Bright Yellow-2 cells and cDNA-amplified fragment length polymorphism-based genomewide expression analysis, we built a comprehensive collection of plant cell cycle-modulated genes. Approximately 1,340 periodically expressed genes were identified, including known cell cycle control genes as well as numerous unique candidate regulatory genes. A number of plant-specific genes were found to be cell cycle modulated. Other transcript tags were derived from unknown plant genes showing homology to cell cycle-regulatory genes of other organisms. Many of the genes encode novel or uncharacterized proteins, indicating that several processes underlying cell division are still largely unknown. Essential genes controlling cell cycle progression have been characterized in different organisms. Recently, genomewide expression analysis in yeast (1) and human cells (2) using microarrays has enlarged the collection of cell cycle-modulated genes to several hundred genes with known and unknown functions. Their transcriptional regulation is strict, and genes involved in the same biological process are most often coexpressed. In plants, the basic regulatory mechanisms controlling cell cycle progression also have been studied (3). Although the core cell cycle genes are conserved among higher eukaryotes, basic developmental differences between plants and other organisms imply that plant-specific regulatory pathways exist that control cell division (4). Especially for events occurring at mitosis, plants are thought to have developed unique mechanisms regulating karyo- and cytokinesis. A typical plant cell is surrounded by a rigid wall and cannot, as such, divide by constriction. Instead, a new cell wall between daughter nuclei is formed by a unique cytoskeletal structure called the phragmoplast, whose position is dictated by another cytoskeletal array called the preprophase band (5). Another major difference between plant and animal mitosis is found in the structure of the mitotic spindles: in animals they are tightly centered at the centrosome, whereas in plants they have a diffuse appearance (6). To identify plant genes involved in cell division and control of cell cycle progression, we performed a genomewide expression analysis of cell cycle-modulated genes in the tobacco Bright Yellow-2 (BY2) cell line. This unique cell line can be synchronized to high levels with different types of inhibitors of cell cycle progression (7, 8). Because of the lack of extensive molecular resources such as genomic sequences, cDNA clones, or ESTs for tobacco, a microarray-based approach cannot be used for transcriptome analysis. Therefore, we used the cDNA-amplified fragment length polymorphism (AFLP) technology to identify and characterize cell cycle-modulated genes in BY2. cDNA-AFLP is a sensitive and reproducible fragment-based technology that has a number of advantages over other methods for genomewide expression analysis (9): it does not require prior sequence information, it allows the identification of novel genes, and it provides quantitative expression profiles. After a detailed analysis, we found that ≈10% of the transcripts are periodically expressed, in agreement with the results obtained in yeast (1). This comprehensive collection of plant cell cycle-modulated genes provides a basis for unraveling the basic mechanisms underlying the plant cell cycle.