Effects of calcitonin gene-related peptide (CGRP) and interleukin 6 (IL-6) on muscle cell differentiation were studied using cultured rat myoblasts (L6 cells). Cell morphology and the amounts of the messenger RNAs (mRNAs) of myogenin and Myf-5, DNA content, creatine kinase (CK) activity, and myoglobin (Mb) content in the cultured cells were examined serially over 10 days of culture. In the presence of CGRP or IL-6, the mRNAs of myogenin and Myf-5 were expressed earlier and at a higher concentration in the treated cells than in the control cells. The ratios of CK activity to DNA content (CK/DNA) and of Mb content to DNA content (Mb/DNA) on day 10 of culture also were greater than in the control cells. Furthermore, the mRNAs of myogenin and Myf-5 in cultured cells incubated with both CGRP and IL-6 increased more rapidly than in cells cultured with CGRP or IL-6 alone, and the ratios of CK/DNA and Mb/DNA on day 10 were more than twice those in the presence of CGRP or IL-6. These findings indicate that both CGRP and IL-6 facilitate the differentiation of myoblasts and may have an additive effect.