The pathway of rhodamine 123 was examined after injection into Sarcophaga flies and after in vitro labeling of the Malpighian tubules. After in vitro labeling the primary cells only retained this potential-sensitive dye for a short period while all secondary cells accumulated the dye from the tubule lumen. In vivo the secondary cells also accumulated rhodamine 123 from the lumen, but the primary cells in the distal parts of all four tubules retained the dye for prolonged periods. This was most pronounced in the distal part of the anterior Malpighian tubules, where rhodamine 123 was eventually precipitated on the luminal concretions. Rhodamine 123 initially accumulated in the secondary cell mitochondria and eventually in intensely fluorescing vesicles, probably lysosomes. No evidence for endocytotic processes from the lumen was found using Lucifer Yellow CH, fluorescent dextrans and fluorescent albumin. Prior incubation with the ionophores valinomycin, nigericin, CCCP (all 1-mu-g/ml), dinitrophenol (1 mM) and NaN3 (10(-2) M) inhibited the selective accumulation of rhodamine 123 to a large extent while monensin (1-5-mu-g/ml) showed little inhibitory effect. Furthermore, only cationic and no anionic or neutral dyes were accumulated by the secondary cells. In the fleshfly Calliphora and the fruitfly Drosophila, the dye rhodamine 123 also selectively accumulated in the secondary cells, as well in vitro as in vivo.