The serotonin (5-HT) 5-HT7 receptor subtype is thought to mediate a number of physiological effects in mammalian brain and periphery. Previous studies suggested that alternative splicing might contribute to 5-HT7 receptor diversity as well. We now report that alternative splicing in human and rat tissues produces four 5-HT7 receptor isoforms that differ in their predicted C-terminal intracellular tails. Human and rat partial 5-HT7 cDNAs and intronic sequences were identified and compared. In rat tissues, three 5-HT7 isoforms, here called 5-HT7(a), 5-HT7(b), and 5-HT7(c), are found. Rat 5-HT7(a) [448-amino acid (aa)] and 5-HT7(b) (435-aa) forms arise from alternative splice donor sites. A third new isoform found in rat, 5-HT7(c) (470-aa), results from a retained exon cassette. Three 5-HT7 mRNA isoforms were also identified in human tissues, where only one isoform was previously described. Two human isoforms represent 5-HT7(a) and 5-HT7(b) forms (445- and 432-aa), but the third form does not correspond to 5-HT7(c). Instead, it constitutes a distinct isoform, 5-HT7(d) (479-aa), resulting from retention of a separate exon cassette. 5-HT7(d) transcripts are not present in rat because the 5-HT7(d)-specifying exon is absent from the rat 5-HT7 gene. A frame-shifting homologue of the rat 5-HT7(c)-Specifying exon is present in the human gene but is not used in the human tissues examined. Tissue-specific splicing differences are present in human between brain and spleen. These studies suggest that alternative splicing may contribute to diversity of 5-HT7 receptor action and that the human and rat repertoires of 5-HT7 splice variants are substantially different.