The transport of a fluorescent cyclosporin analogue was measured in killifish (Fundulus heteroclitus) proximal tubules by means of epifluorescence microscopy and digital image analysis. Renal cells rapidly accumulated the cyclosporin analogue from the medium and attained steady state within 60 min; luminal fluorescence increased over the first 60-90 min. At steady state, luminal fluorescence intensity was two to three times higher than cellular. Cellular fluorescence intensity was a linear function of medium substrate concentration and was not affected by any treatment used. In contrast, luminal fluorescence exhibited a saturable component as the medium concentration of the cyclosporin was increased. Secretion into the lumen was blocked by metabolic inhibitors, vanadate, other cyclosporins, such as cyclosporin A and cyclosporin G, and substrates for P-glycoprotein (verapamil, vinblastine, and quinine) but not by substrates for the renal organic anion or organic cation transport systems, such as p-aminohippurate or tetraethylammonium. The data are consistent with the fluorescent cyclosporin analogue entering proximal tubule cells by simple diffusion and then being pumped into the tubular lumen by P-glycoprotein.