Previous work suggested that the cocaine analog [(125)I]RTI-55 labels a novel binding site in rat brain membranes, which is not associated with the dopamine (DA), serotonin (5-HT), or norepinephrine (NE) transporters [Rothman et al. 1995 J Pharmacol Exp Ther 274:385-395]. Here, we tested whether this site is a product of the DA transporter (DAT) gene. We used a T-antigen knock-in at the DAT gene that results in an effective DAT knock-out (KO) confirmed by Southern blot, DAT immunohistochemistry, and [(125)I]RTI-55 ligand binding. Brain membranes were prepared from frozen whole brain minus caudate of wild-type (WT) B6/Sv129, +/+ and minus sign/minus sign (KO) mice. KO mice were used at approximately 23 days of age. Binding surface analysis of [(125)I]RTI-55 binding to membranes prepared from the brains of WT mice, with 100 nM citalopram to block binding to the 5-HT transporter (SERT), revealed two binding sites: the DAT and a second site, replicating previous studies conducted with rat brains. In the absence of the DAT (minus sign/minus sign mice), binding surface analysis demonstrated that [(125)I]RTI-55 labeled two sites: the NET and a second site called site "X." Structure-activity studies of site "X" demonstrated that high-affinity ligands for the DAT, NET, and SERT have low or negligible affinity for site "X." The relatively high density of site "X" in brain membranes and the fact that the K(i) values of cocaine and cocaethylene for site "X" are in the range achieved in the brain following cocaine administration suggests that site "X" could contribute to the pharmacological or toxicological effects of cocaine. Further progress in delineating the function of site "X" will depend on developing potent and selective agents for this site.