An asymmetric fourth cell division in the sea urchin embryo results in formation of daughter cells, macromeres and micromeres, with distinct sizes and fates. Several lines of functional evidence presented here, including pharmacological interference and dominant negative protein expression, indicate that heterotrimeric G protein Gi and its interaction partner, activator of G-protein signaling (AGS), are necessary for this asymmetric cell division. Inhibition of Gi signaling by pertussis toxin interferes with micromere formation and leads to defects in embryogenesis. AGS was isolated in a yeast two-hybrid screen with G alpha i as bait and was expressed in embryos localized to the cell cortex at the time of asymmetric divisions. Introduction of exogenous dominant-negative AGS protein, containing only G-protein regulatory (GPR) domains, selectively prevented the asymmetric division in normal micromere formation. These results support the growing evidence that AGS is a universal regulator of asymmetric cell divisions in embryos.