Serotonin (5-HT7) receptor pre-mRNA is alternatively spliced in rat tissue to produce three isoforms, 5-HT(7a), 5-HT(7b) and 5-HT(7c), which differ in the amino acid sequences of their carboxyl terminal tails. Substantial species differences in structure and expression patterns exist for 5-HT7 isoforms. We have now compared some of the functional characteristics and level of expression for the three rat 5-HT7 receptor isoforms. Recombinant receptor isoforms were expressed in COS-7 cells for examination of [3H]5-HT binding characteristics and in JEG-3 cells to ascertain their ability to stimulate cAMP production. These studies showed that all three isoforms are functionally active and have similar agonist binding characteristics. Distribution of expression of the three rat receptor isoforms were examined in several brain regions and peripheral tissues using RT-PCR and in situ hybridization. The relative proportions of total 5-HT7 receptor message lent by each isoform varied little between these areas. In contrast to what has been observed in human tissue, the 5-HT(7a) isoform predominated in all regions examined, while the 5-HT(7c) isoform revealed a low level of expression (3% of total transcript). In situ hybridization was used to determine if the overall low level of expression of the 5-HT(7c) isoform by RT-PCR could be attributed to a small localized subpopulation of cells expressing high levels 5-HT(7c) message. In situ hybridization results indicate a generalized low level of expression of the 5-HT(7c) isoform throughout the CNS. These data suggest that while all three known 5-HT7 receptor isoforms in the rat are functionally competent, any functionally important differences between the three isoforms are not likely to involve differences in ligand binding or gross differences in adenylate cyclase coupling. However, differences in receptor phosphorylation, regulation or coupling to other effectors or cell trafficking could still exist.