This study describes a direct comparison of dopamine transporter (DAT) mRNA and protein, as well as its binding sites, in tissue from the same animals after chronic cocaine administration. Rats were treated twice daily with 25 mg/kg cocaine or with saline. After 8 days of cocaine administration, changes in DAT mRNA levels in the substantia nigra pars compacta and ventral tegmental area were measured by in situ hybridization, and DAT protein in the striatum was quantified by immunoblotting. Whereas chronic cocaine treatment significantly reduced levels of DAT mRNA in the substantia nigra pars compacta and ventral tegmental area as compared with vehicle-treated controls, cocaine treatment did not alter DAT protein levels in the striatum. Furthermore, the density of DAT binding sites was also measured in the striatum by quantitative autoradiography using two DAT radioligands, 33-(4-[125I]iodophenyl)tropane-2-carboxylic acid methyl ester ([125I]RTI-55) and [3H]propanoyl-3beta-(4-tolyl)tropane ([3H]PTT). Similar to the results of immunoblotting of DAT protein, RTI-55 and [3H]PTT binding site levels also remained unaltered. These results indicate a dissociation in the regulation of DAT mRNA and its protein levels as a result of cocaine administration in rats. This study also indicates that the DAT ligands [3H]PTT and [125I]RTI-55 provide an accurate assessment of DAT protein levels.