Isolated rat renal cortical tubules were used to study the nature of homocystine entry into the tubule cell and its transport interactions with cystine and the dibasic amino acids. The uptake of homocystine with time was progressive, reaching a steady state after 60 min. of incubation. Analysis of the intracellular pool after 5 and 30 min. of incubation revealed that virtually all of the transported homocystine had been converted to other metabolites of the transsulfuration pathway. The major metabolite was cystathionine with a somewhat lesser, but still significant amount as S-adenosylhomocysteine. A kinetic analysis showed that two systems for cellular entry of homocysteine existed with a Km1 of 0.17 mM and a Km2 of 7.65 mM. Arginine and lysine inhibited homocystine uptake via the low Km, high affinity system, but appeared not to inhibit the high Km, low affinity system. Cystine inhibited the low Km, high affinity system, but had an indeterminate effect on the high Km, low affinity system. Homocystine inhibited the uptake of cystine, lysine and arginine by isolated rat renal cortical tubules. The inhibition of homocystine on cystine uptake appeared to occur on both the high and low Km system for tubule cell entry of cystine. The data suggest that the low Km system for homocystine transport is shared with cystine and the dibasic amino acids. These data extend the knowledge of homocystine metabolism and provide a rational basis for new approaches to the treatment of homocystinuria.