Drosophila Scribble is implicated in the development of normal synapse structure and epithelial tissues, but it remains unclear how it plays a role and which process it controls [1–3]. The mammalian homolog of Scribble, hScrib, has a primary structure and subcellular localization similar to that of its fly homolog [4], but its function remains unknown. Here we have used tandem mass spectrometry to identify major components of the hScrib network. We show that it includes βPIX (also called Cool-1), a guanine nucleotide exchange factor (GEF), and its partner GIT1 (also called p95-APP1), a GTPase activating protein (GAP) [5–8]. βPIX directly binds to the hScrib PDZ domains, and the hScrib/βPIX complex is efficiently recovered in epithelial and neuronal cells and tissues. In cerebellar granule cell cultures, hScrib and βPIX are both partially localized at neuronal presynaptic compartments. Furthermore, we show that hScrib is required to anchor βPIX at the cell cortex and that dominant-negative βPIX or hScrib proteins can each inhibit Ca²⁺-dependent exocytosis in neuroendocrine PC12 cells, demonstrating a functional relationship between these proteins. These data reveal the existence of a tight hScrib/βPIX interaction and suggest that this complex potentially plays a role in neuronal transmission.