rCAT3 (rat cationic amino acid transporter 3), a cDNA that encodes a novel member of the murine CAT family was isolated. The protein encoded by rCAT3 contained 619 amino acids, 53-58% of which were identical with those of the murine CAT family proteins previously described (mouse CAT1, CAT2a, CAT2b, and rat CAT1). Transient expression of rCAT3 and L-[14C]arginine incorporation experiments in COS7 cells verified a high affinity system y+ transporter activity of rCAT3. First, rCAT3-mediated L-[14C]arginine incorporation was time-dependent and saturable with half-saturation constant (Km) values of 103 +/- 12 microM (mean +/- S.E., n = 3). Second, the incorporation was specific for cationic amino acids as evidenced from the inhibition by L-arginine, L-lysine, and L-ornithine. Third, neither sodium nor chloride ions in the extracellular medium were required for the activity. Fourth, the incorporation was inhibited by high potassium-induced membrane depolarization. On Northern blot using RNAs from various rat tissues, the expression of rCAT3 mRNA was restricted to the brain. These results indicated a role of rCAT3 in the system y+ transporter activity in the nervous tissue.