K252a, an indrocarbazole derivative and protein kinase inhibitor, is reported to promote myogenic differentiation in C2 mouse myoblasts. We examined the effects of K252a on QM-RSV cells, quail myoblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus. K252a promoted myotube formation of QM-RSV cells. Presumptive QM-RSV cells also formed multinucleated cells when exposed to K252a. However, the expression of myogenin, a muscle regulatory factor, was not stimulated in the presence of the drug, suggesting that it promotes membrane fusion but not myogenic differentiation. To confirm the promotion of membrane fusion by K252a, presumptive C2 cells, which are strongly resistant to HVJ-mediated cell fusion, were fused by HVJ (Sendai virus) after K252a treatment. Presumptive C2 cells treated with K252a fused with HVJ, demonstrating that K252a causes the cells to enter a fusion-capable state. The amount of membrane cholesterol, a factor that decreases membrane fluidity, fell in K252a-treated C2 cells. The results suggest that a decrease of membrane cholesterol is a cause of the change that renders myoblast membrane susceptible to fusion in the presence of K252a.