Multidomain guanine nucleotide (GDP/GTP) exchange factor (GEF) proteins coordinate diverse inputs that signal the actin cytoskeleton. Mammals have two such proteins (Kalirin, Trio), while Drosophila has one, which plays essential roles within and outside the nervous system. For Kalirin, numerous isoforms containing different combinations of functional domains are generated through alternative splicing and use of alternative transcriptional start sites. These different isoforms potentially allow a wide variety of proteins to interact with Kalirin, thereby affecting the activity of the functional domains. Humans, like rats, express a large set of Kalirin isoform mRNAs, and we identified a novel Kalirin isoform, containing only the second GEF domain. Kalirin isoforms are predominantly expressed in the brain, while Trio is expressed in a wider variety of tissues. Alternative splicing and transcription of Kalirin are differentially regulated during development in rats and humans, resulting in expression of isoforms of Kalirin containing different functional domains at different times and locations. The prevalence of Kalirin in the cortex throughout life suggests roles in axonal development and the mature brain.