Amphiphysin, an SH3-domain containing protein concentrated in nerve terminals, is believed to be involved in the synaptic vesicle recycling. We have cloned cDNAs of a novel isoform of amphiphysin (amphiphysin II) by exploiting sequence information for homologous ESTs deposited in databases. At least 9 different subtypes of the isoform with 50-60% amino acid identity to the human amphiphysin were identified by a conventional library screening and PCR amplification of cDNA libraries. Each subtype probably represents a splice variant of a single gene transcript. Analysis of mRNA expression in various tissues by RT-PCR showed that the isoform is ubiquitously distributed. The expression spectrum of the isoform subtypes, however, is significantly different in several tissues examined, suggesting that they are involved in the regulation of endocytic processes that are unique to each cell type.