Proliferation of aortic smooth muscle cells contributes to atherogenesis and neointima formation. Sublytic activation of complement, particularly C5b-9, induces cell cycle progression in aortic smooth muscle cells. RGC-32 is a novel protein that may promote cell cycle progression in response to complement activation. We cloned human RGC-32 cDNA from a human fetal brain cDNA library. The human RGC-32 cDNA encodes a 117-amino acid protein with 92% similarity to the rat and mouse protein. Human RGC-32 maps to chromosome 13 and is expressed in most tissues. Sublytic complement activation enhanced RGC-32 mRNA expression in human aortic smooth muscle cells and induced nuclear translocation of the protein. RGC-32 was physically associated with cyclin-dependent kinase p34CDC2 and increased the kinase activity in vivo and in vitro. In addition, RGC-32 was phosphorylated by p34CDC2-cyclin B1 in vitro. Mutation of RGC-32 protein at Thr-91 prevented the p34CDC2-mediated phosphorylation and resulted in loss of p34CDC2 kinase enhancing activity. Overexpression of RGC-32 induced quiescent aortic smooth muscle cells to enter S-phase. These data indicate that cell cycle activation by C5b-9 may involve p34CDC2 activity through RGC-32. RGC-32 appears to be a cell cycle regulatory factor that mediates cell proliferation, both as an activator and substrate of p34CDC2.