The actions of the thyroid hormone (T3) are modified by other hormones. Therefore, in the normal cell culture system, with serum as a medium ingredient, it is difficult to eliminate the influences of other hormones derived from the serum. In the present study, two experiments were conducted to clarify the effects of T3 on differentiation, growth and proteolysis (experiment 1), and concerted effects of T3 and insulin (experiment 2) in a serum-free culture with the use of muscle cells originated from chick embryo. Protein content and creatine kinase (CK) activity were examined as indices of growth and differentiation of the muscle cells, respectively, and N(tau)-methylhistidine (MeHis) release was examined as an index of myofibrillar proteolysis. It was observed that T3 suppressed both the muscle differentiation and proteolysis in the serum-free medium (experiment 1), though in our previous experiment they were enhanced by T3 in the serum-supplemented normal medium. On the other hand, T3 increased myofibrillar proteolysis and had no effect on muscle differentiation when insulin was included in the serum-free medium (experiment 2). These results clearly show that the effects of thyroid hormones on muscle differentiation and proteolysis are apparently different when serum is deprived from the medium, and these differential effects of thyroid hormone could be partially explained by an interaction with insulin, one of the growth factors in the serum.