Pix, a Pak-interacting exchange factor, is known to be involved in the regulation of Cdc42/Rac GTPases and Pak kinase activity. In this study, we cloned the cDNAs encoding two βPix isoforms from mouse brain cDNA library. Both of the cloned genes, designated βPix-b and βPix-c (GenBank Accession Nos. AF247654 and AF247655, respectively), have a novel insert region consisting of 59 amino acid residues. In βPix-c, 75 amino acid residues are deleted in the proline-rich region at the carboxyl-terminus of βPix. In situ hybridization studies with insert region-specific probe in rat embryo show that insert region-containing isoforms are expressed mainly in the central nervous system. Moreover, temporal expression pattern of isoforms is correlated with the active neurogenesis period in the cerebral cortex and cerebellum. These results strongly suggest that βPix isoforms may play important roles in the cellular events required for brain development such as neuronal migration.