We found that eosinophil cationic protein (ECP) stimulated the growth of mouse Balb/c 3T3 fibroblasts. ECP-treated 3T3 cells were more flattened and exhibited enhanced stress fiber formation. The enhancement of cytoskeleton after addition of recombinant ECP appeared stable and was able to inhibit disassembly of actin filaments that was induced by fibroblast growth factor-2. The ROCK inhibitor, Y-27632, abrogated this enhancement on stress fiber formation that was induced by ECP indicating the involvement of Rho/ROCK signaling pathway. The effect of ECP was assessed on the differentiation of primary cardiomyocytes derived from rat neonatal heart since the development of actin filaments is significantly related with organization of stress fibers. As the result, both beating rate and the expression of cardiac muscle specific markers such as atrial natriuretic factor were enhanced in the presence of ECP. Thus ECP may also function as a cardiomyocyte differentiation factor.