Literature reports suggest that nitric oxide (NO) participates in the regulation of dopaminergic neurotransmission, possibly through interaction with cysteine residues of the dopamine transporter (DAT). Rotating disk electrode voltammetry was used to measure dopamine (DA) transport in rat striatum to determine if 1) the nitric oxide synthase (NOS) substrate, L-arginine (L-Arg), could affect DAT activity; 2) L-Arg-dependent effects on DAT activity could be blocked by NOS and guanylate cyclase inhibitors, a NO scavenger, DA, and cocaine; 3) a NO donor could affect DAT activity; and 4) L-Arg could protect the DAT from a sulfhydryl agent. L-Arg increased DAT activity by increasing V(max). NOS inhibitors (S-ethylisothiourea and S-isopropylisothiourea), a NO scavenger (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide), DA, and cocaine blocked the L-Arg effect. The guanylate cyclase inhibitor, 1H-(1,2,4)-oxadiazolo[4,3a]quinoxalin-1-one, did not. The NO donor, S-nitroso-N-acetylpenicillamine, decreased DAT activity and L-Arg protected the DAT from the effects of the sulfhydryl agent N-ethylmaleimide. These results suggest that L-Arg, via NO, may play a role in regulating DAT activity in rat striatum by increasing the V(max) of DA transport. Furthermore, it is suggested that the effects of L-Arg on DAT activity may be due to modification of the DAT itself, possibly via the NO-mediated modification of DAT cysteine residues. Finally, NO produced from L-Arg may affect the DAT differently than NO from NO donors. These results further the notion that dopaminergic neurotransmission may be regulated by changes in DAT activity caused by L-Arg and NOS.