The purpose of this study was to analyze the transcriptional regulation of the zebrafish solute carrier family 6 member 3 gene (slc6a3, dopamine transporter, dat), as a first step towards isolating regulatory sequences useful for driving transgene expression within dopaminergic neurons of the zebrafish CNS in vivo. We found that the 3.0 kb slc6a3 mRNA is expressed in each of the major groups of dopaminergic neurons previously identified in the zebrafish CNS. The slc6a3 gene spans >20 kb of genomic DNA and contains 15 exons. The genomic organization of slc6a3 is highly conserved with respect to its human orthologue, including the presence of an untranslated first exon. The promoter lacks a canonical TATA box and there are multiple transcriptional start sites. Functional analysis of cis-acting elements responsible for the expression pattern of slc6a3 was carried out by generating stable transgenic zebrafish lines expressing fluorescent reporters under transcriptional control of fragments of slc6a3 genomic sequence. The region between -2 kb and +5 kb with respect to the transcriptional start site contains the core slc6a3 promoter, in addition to neuronal enhancers and/or non-neuronal repressors that restrict expression to the CNS, but this region lacks cis-acting elements responsible for slc6a3 expression in dopaminergic neurons. The upstream sequence between -6 kb and -2 kb contains an enhancer element that drives slc6a3 expression in dopaminergic neurons of the pretectal region, and additional sequences that partially repress expression in non-dopaminergic neurons. However, expression of slc6a3 in dopaminergic neurons of the ventral diencephalon and telencephalon is dependent on elements that lie outside the region -6 kb to +5 kb. These data provide a detailed analysis of the slc6a3 gene and show that its expression in different populations of dopamine neurons is driven by discrete enhancers, rather than a single target sequence for a terminal factor involved in specifying neurochemical phenotype.