Transformer splicing regulatory proteins determine the sexually dimorphic traits of Drosophila. The role of the vertebrate homologs of Tra-2 in phenotypic specification is undefined. We are using the alternative splicing of the MYPT1 E23 exon as a model for the study of smooth muscle diversification into fast and slow contractile phenotypes. Tra2 mRNA and protein is expressed at up to 10-fold higher levels in fast smooth muscle tissues such as the rat portal vein and small mesenteric artery, in which E23 is spliced, as compared to the slow smooth muscle tissues of the large arteries and veins, in which E23 is skipped. Tra2 is upregulated up to 10-fold concordant with the initiation of E23 splicing as the rat portal vein and avian gizzard implement the fast program of gene expression in the perinatal period. In disease models such as portal hypertension and mesenteric artery high/low flow, the portal vein and first order mesenteric artery dynamically downregulate Tra2 concordant with a shift to E23 skipping and the slow program of gene expression. Tra2 binds to a highly conserved sequence within E23 and transactivates its splicing in vitro and in vivo; this is abolished with mutation or deletion of this sequence. RNA interference–mediated knockdown of Tra2 markedly reduces E23 splicing. We propose that Tra2 has been conserved through evolution and redeployed for the specification of the fast smooth muscle phenotype and may serve as a novel nodal point for the investigation of this process in developmental and disease models.