Effects of in vivo, intrastriatal administration of RTI-76 ¿3 beta-(3-p-chlorophenyl) tropan-2 beta-carboxylic acid p-isothiocyanato-phenylethyl ester hydrochloride¿, an irreversible inhibitor of dopamine transporter (DAT) binding in vitro, on [125I]RTI-55 ¿3 beta-[4-iodophenyl]tropan-2 beta-carboxylic acid methyl ester tartrate¿ binding to striatal DAT in vitro were examined in male rats. Effects on [3H]DAT and D1 dopamine receptor binding in vitro after intrastriatal RTI-76 injection were also determined. One hour after direct intrastriatal injection, RTI-76 caused a dose-related increase in KD for [125I]RTI-55 binding in vitro in striatal tissue, without affecting transporter maximum binding (Bmax). In contrast, 24 hr after administration, RTI-76 caused a dose-related decrease in striatal DAT Bmax without affecting KD, a decrease that reversed over the next several days. Transport of [3H]dopamine into synaptosomes was decreased similarly. Intrastriatal injection of reversible inhibitors of DAT, such as cocaine or WIN-35428 ¿3 beta-[4-fluorophenyl]tropan-2 beta-carboxylic acid methyl ester tartrate), was without effect on transporter binding 1 and 6 days after administration. RTI-76 had little effect on [3H]SCH-23390 ¿R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4, 5-tetrahydro-1H-3-benzazepine¿ binding 1 or 24 hr after intrastriatal injection, indicating at least some selectivity of RTI-76 for DAT. The RTI-76-induced decrease in Bmax, as well as the concurrent decrease in [3H]DAT, were reversible, with the T1/2 of transporter recovery estimated to be 6 days.