Ca(2+)/calmodulin (CaM)-dependent protein kinase I (CaM-KI), which is a member of the multifunctional CaM-K family, is thought to be involved in various Ca(2+)-signalling pathways. In this report, we demonstrate that CaM-KI activated by an upstream kinase (CaM-K kinase), but not unactivated CaM-KI, phosphorylates myosin II regulatory light chain (MRLC) efficiently ( K (cat), 1.7 s(-1)) and stoichiometrically (approximately 0.8 mol of phosphate/mol) in a Ca(2+)/CaM-dependent manner in vitro. One-dimensional phosphopeptide mapping and mutational analysis of MRLC revealed that the activated CaM-KI monophosphorylates only Ser-19 in MRLC. Transient expression of the Ca(2+)/CaM-independent form of CaM-KI (CaM-KI(1-293)) in HeLa cells induced Ser-19 phosphorylation of myosin, II accompanied by reorganization of actin filaments in the peripheral region of the cells. CaM-KI-induced reorganization of actin filaments was suppressed by co-expression of non-phosphorylatable MRLC mutants (S19A and T18AS19A). Furthermore, a kinase-negative form of CaM-KI (CaM-KI(1-293,K49E)) significantly reduced reorganization of actin filaments, indicating a dominant negative effect. This is the first demonstration that the activation of the CaM-KI cascade induces myosin II phosphorylation, resulting in regulation of actin filament organization in mammalian cells.