A small subset of neurons in the nematode Caenorhabditis elegans utilizes the catecholamine dopamine (DA) as a neurotransmitter to control or modulate movement and egg-laying. Disruption of DA-mediated behaviors represents a potentially powerful strategy to identify genes that are likely to participate in dopaminergic systems in man. In vertebrates, extracellular DA is inactivated by presynaptic DA transport proteins (DATs) that are also major targets of addictive agents, including amphetamines and cocaine. We used oligonucleotides derived from the C. elegans genomic locus T23G5.5 to isolate and characterize T23G5.5 cDNAs. Our studies predict that mRNAs from this locus encode a 615-amino-acid polypeptide with twelve stretches of hydrophobicity suitable for transmembrane domains, similar to that found in vertebrate catecholamine transporters. The inferred translation product bears highest identity (43-47%) to catecholamine (DA, norepinephrine, epinephrine) transporters within the GAT1/NET gene family and possesses conserved residues implicated in amine substrate recognition. Consistent with these findings, HeLa cells transfected with the C. elegans cDNA exhibit saturable and high affinity DA transport (Km = 1.2 microM) that is dependent on extracellular Na+ and Cl- and blocked by inhibitors of mammalian catecholamine transporters, including norepinephrine transporter- and DAT-selective antagonists, tricyclic antidepressants, and the nonselective amine transporter antagonists cocaine and D-amphetamine. These studies validate the T23G5.5 locus as encoding a functional catecholamine transporter, providing important comparative sequence information for catecholamine transporter structure/function studies and a path to identify regulators of dopaminergic signaling via genetic or pharmacologic manipulation of C. elegans cDNA in vivo.