A novel D3 receptor cDNA containing an insertion of 84 bp has been isolated from a series of alternatively spliced D3 receptor cDNAs by polymerase chain reaction on RNA isolated from rat basal forebrain. Sequence analysis of both subcloned cDNAs and genomic DNA clones, has identified a 84 bp insertion resulting in a 28 amino acid insertion in the first extracellular loop. A comparison of the cDNA sequence of the novel D3 cDNA and the isolated genomic sequence indicates that the novel insertion corresponds to a new exon and arises via an alternative splicing event. The mRNA for the novel insertion could be detected by PCR in several areas of the brain co-localized with the wild type form of the D3 receptor. Moreover, genomic Southern blot analyses suggest that the D3-receptor exon giving rise to this novel D3 mRNA variant might exist in other species.