Nitric oxide (NO), a known relaxant, is produced in cells from L-arginine (L-Arg). Because the relaxation of retinal pericytes alters the microcirculatory hemodynamics, it is important to understand the manner of NO production in retinal pericytes. The purpose of this study was to clarify the molecular mechanism(s) of uptake of L-Arg in retinal pericytes using a conditionally immortalized rat retinal pericyte cell line (TR-rPCT1 cells) which expresses the mRNAs of endothelial NO synthase and inducible NO synthase. L-Arg uptake by TR-rPCT1 cells exhibited Na⁺-independence and concentration-dependence with a K _m of 28.9 µM. This process was strongly inhibited by substrates of cationic amino acid transporters (CAT), such as L-ornithine and L-lysine. In contrast, L-valine, L-leucine, and L-glutamine, which are substrates of cation/neutral amino acid transport systems, such as system y⁺L, system B^0,+, and system b^0,+, did not strongly inhibit L-Arg uptake by TR-rPCT1 cells. In addition, the expression of mRNA and protein of CAT1 in TR-rPCT1 cells was observed by reverse transcription-polymerase chain reaction and immunoblot analyses. Taking these results into consideration, it appears that CAT1 is involved in L-Arg uptake by retinal pericytes and this is expected to play an important role in the relaxation of retinal pericytes, thereby modulating the microcirculatory hemodynamics in the retina.