CaMK-II (the type II multifunctional Ca2+/calmodulin kinase) is a ubiquitous serine/threonine protein kinase that is activated by Ca2+ and calmodulin (CaM) and has been implicated in cell cycle control. NIH 3T3 fibroblast cytosolic extracts contain CaMK-II enzymatic activity and two major Ca2+/CaM-dependent phosphoproteins of M(r) 55,000 and 115,000. Reverse transcription-PCR indicates that the gamma B and gamma C isozymes of CaMK-II are predominately expressed. KN-93, a novel membrane-permeant synthetic inhibitor of purified neuronal CaMK-II, inhibits serum-induced fibroblast cell growth in a comparable dose-dependent fashion to its inhibition of CaMK-II activity. After 2 days of KN-93 treatment, 95% of cells are arrested in G1. G1 arrest is reversible; 1 day after KN-93 release, a peak of cells had progressed into S and G2-M. KN-92, a similar but inactive compound, had no effect on CaMK-II activity or cell growth. KN-93 also blocked cell growth stimulated by basic fibroblast growth factor, platelet-derived growth factor-BB, epidermal growth factor, and insulin-like growth factor-1. After 3 days of KN-93-induced G1 arrest, cell size and viability decreased and DNA fragmented, indicating apoptosis. These data suggest that CaMK-II is necessary for cell cycle progression through G1 and operates at a site common to the transduction of signals from growth and/or survival factors.