Unidirectional fluxes of L-35S-cystine and intracellular 35S activity were measured in isolated perfused segments of rabbit proximal straight tubule. The absorptive (lumen-to-both) flux of L-35S-cysteine showed a tendency toward saturation within the concentration limits imposed by the low solubility of cystine (0.3 mmol . l-1). In contrast, for the bath-to-lumen fluxes, there was a linear relation between the bathing solution concentration of L-35S-cystine and the rate of 35S appearance in the lumen. Nonlinear fitting of both sets of unidirectional flux data gave a maximal cystine transport rate (Jmax) of 1.45 +/- 0.27 (SEM) pmol min-1 mm-1, a Michaelis constant (Km) of 0.20 +/- 0.07 mmol . l-1, and an apparent permeability coefficient of 0.27 +/- 0.11 pmol min-1 mm-1 (mmol . l-1)-1 (approximately 0.06 micrometer/s). The 35S concentration in the cell exceeded that in the lumen by almost 60-fold during the lumen-to-bath flux, and exceeded the bathing solution concentration by 4.7-fold during the bath-to-lumen flux. Thus cystine was accumulated by the cells across either membrane, but over 77% of the intracellular activity was in the form of cysteine. Although the presence of luminal L-lysine or cycloleucine inhibited the absorptive flux of cystine, neither amino acid affected the bath-to-lumen flux.