Cystatin-related proteins (CRPs) are abundant androgen-regulated secretory glycoproteins that are specifically synthesized in the ventral prostate and lachrymal gland of the rat. Two complete 6-kb genes, Crp1 and Crp2, have been cloned and characterized. They are differentially expressed and encode slightly different proteins. The genes each contain four exons which are interrupted by large introns. An alignment of their sequences demonstrates an overall homology of 90%. The 3' end of a third gene, Crp3, from which only a 1.5-kb fragment was isolated, displays a sequence identity of 84%. These data indicate the existence of a Crp multigene family. The 5' flanking regions of Crp1 and Crp2 are highly homologous and contain a GATAAA sequence 29 nt upstream from the transcription start point. This TATA-box-like element is also found in the promoters of the genes encoding cystatin type-2 proteins. No other recognizable transcription control elements can be detected. Potential binding sites (ARE) for the androgen receptor are scattered throughout the entire genes. The exon/intron organization of the genes encoding CRPs, the size of the exons and their encoding amino acid sequences exhibiting a characteristic spacing of the Cys residues are structural elements displaying a remarkable similarity with the corresponding elements in the genes encoding cystatin type-2 proteins. CRPs must therefore belong to the cystatin superfamily. However, due to their additional domain encoded in an extra exon 2, CRPs must be classified as a new family, type 5.