We report here the molecular cloning of three new splice variants of the human serotonin 5-hydroxytryptamine4 (h5-HT4) receptor, which we named h5-HT4(b), h5-HT4(c), and h5-HT4(d). The sequence following the splicing site at Leu358 in the C-terminal tail of h5-HT4(b) displays a 74% protein identity with the same region in the long form of the rat 5-HT4 receptor (r5-HT4L) but is shorter by 18 amino acids compared to its rat counterpart. The splice variants h5-HT4(c) and h5-HT4(d) are the first of their kind to be described in any animal species. The C terminus of h5-HT4(c) displays a high number of putative phosphorylation sites. The h5-HT4(d) isoform corresponds to an ultrashort form of the receptor, with a truncation two amino acids after the splicing site. Tissue distribution studies revealed some degree of specificity in the pattern of expression of the different isoforms within the human body. The four splice variants transiently expressed in COS-7 cells displayed an identical 5-HT4 pharmacological profile and showed a similar ability to stimulate adenylyl cyclase activity in the presence of 5-HT. The stimulatory pattern of cyclic AMP formation in response to the 5-HT4 agonist renzapride was found to be significantly different between h5-HT4(a) and the other h5-HT4 isoforms, indicating that the splice variants may differ in the way they trigger the signal transduction cascade following receptor activation.