The activation of sea urchin eggs at fertilization provides an ideal system for studying the molecular events involved in cellular activation. Rho GTPases, which are key signaling enzymes in eukaryotes, are involved in sustaining the activation of sea urchin eggs; however, their downstream effectors have not yet been characterized. In somatic cells, RhoA regulates a serine/threonine kinase known as Rho-kinase (ROCK). The activity of ROCK in early sea urchin development has been inferred, but not tested directly. A ROCK gene was identified in the sea urchin (Strongylocentrotus purpuratus) genome and the sequence of its cDNA determined. The sea urchin ROCK (SpROCK) sequence predicts a protein of 158 kDa with >72% and 45% identities with different protein orthologues of the kinase catalytic domain and the complete protein sequence, respectively. SpROCK mRNA levels are high in unfertilized eggs and decrease to 35% after 15 min postfertilization and remain low up to the 4 cell stage. Antibodies to the human ROCK-I kinase domain revealed SpROCK to be concentrated in the cortex of eggs and early embryos. Co-immunoprecipitation assays indicate that RhoA and SpROCK are physically associated. This association is destroyed by treatment with the C3 exoenzyme and with the ROCK antagonist H-1152. H-1152 also inhibited DNA synthesis in embryos. We conclude that the Rho-dependent signaling pathway, via SpROCK, is essential for early embryonic development.