Two independent approaches to understanding the molecular mechanism of cytokinesis have converged on the gene spaghetti-squash (sqh). A genetic screen for mitotic mutants identified sqh1, a mutation that disrupts cytokinesis, which was then cloned by transposon tagging. Independently, the gene that encodes the regulatory light chain of the biochemically defined nonmuscle myosin (MRLC-C) was also cloned. We show here that sqh encodes MRLC-C and that in sqh1 mutants, the level of stable light chain transcript is greatly reduced. Reversion by transposon excision or transformation with a wild-type copy of the sqh transcription unit rescues cytokinesis failure and other defects in sqh1. Vertebrate homologs of MRLC-C are phosphorylatable and regulate myosin activity in vitro. These studies provide genetic proof that MRLC-C is required for cytokinesis, suggest a role for the protein in regulating contractile ring function, and establish a genetic system to evaluate its function.