The mechanisms whereby insulin analogues may cause enhanced mitogenicity through activation of either the IR (insulin receptor) or the IGF-IR (insulin-like growth factor 1 receptor) are incompletely understood. We demonstrate that in L6 myoblasts expressing only IGF-IRs as well as in the same cells overexpressing the IR, IGF-I (insulin-like growth factor 1), insulin and X10 (AspB10 insulin) down-regulate the mRNA expression level of the cell cycle inhibitor cyclin G2, as measured by qRT-PCR (quantitative reverse transcription-PCR), and induce cell growth measured by [6-(3)H]thymidine incorporation into DNA. Western blotting showed a marked down-regulation of cyclin G2 at the protein level in both cell lines. Overexpression of cyclin G2 in the two cell lines diminished the mitogenic effect of all three ligands. The use of specific inhibitors indicated that both the MAPK (mitogen-activated protein kinase) and the PI3K (phosphoinositide 3-kinase) pathways mediate the down-regulation of Ccng2. The down-regulation of CCNG2 by the three ligands was also observed in other cell lines: MCF-7, HMEC, Saos-2, R(-)/IR and INS-1. These results indicate that regulation of cyclin G2 is a key mechanism whereby insulin, insulin analogues and IGF-I stimulate cell proliferation.