Mammalian Trio is a multifunctional, multidomain Rho guanine nucleotide exchange factor (GEF) closely related to Kalirin. Trio is important for proper axon guidance in Drosophila, and mice lacking Trio exhibit both skeletal muscle and neuronal disorders. Full length mammalian Trio and Kalirin both consist of a Sec14P-like domain, several spectrin-like domains, two Rho GEF domains each containing a Dbl-homology (DH) and a pleckstrin-homology (PH) domain, two src homology 3 domains (SH3), Ig/fibronectin-like domains (Ig/FN), and a kinase domain. We have previously described multiple isoforms of Kalirin derived through alternative splicing and multiple transcription start sites, but multiple isoforms of Trio containing different functional domains have not been described. Using a new antibody directed against the spectrin-like region of rat Trio coupled with reverse transcription PCR and cDNA sequencing, we have identified 4 novel isoforms of Trio expressed in rat cortex and cerebellum. Two isoforms, Trio 9S and Trio 9L, are derived through alternative splicing of Trio exon 48 and are abundantly expressed in rat brain. Trio 8 is expressed in postnatal day 30 and adult cerebellum, but not in cortex or skeletal muscle. Trio/duet is expressed in adult cortex and cerebellum. In the rat brain, each of these Trio isoforms is expressed at a higher level than full length Trio.