The effect of 12-O-tetradecanoyl phorbol-13-acetate (TPA) on proliferation and differentiation of the human embryonal rhabdomyosarcoma cell line RD was investigated. The proliferation of RD cells is drastically and reversibly inhibited by 100 nM TPA. The effect is evident after 24 h of treatment and is maximal after 50-70 h. The reduction of proliferation in treated cells is followed by increased expression of differentiative characters such as a large increase in muscle myosin expression and in the binding of 125I-alpha-bungarotoxin. Moreover TPA induces the appearance of myotube-like structures, which contain bundles of thick and thin myofilaments along with Z bodies. The described effects are not observed if the TPA-containing medium is replaced daily, thus suggesting that these effects might be related to substances secreted by treated cells. The phosphorylation of three proteins is significantly stimulated by TPA within minutes of its administration to RD cells. Although with a different pattern, the stimulation of protein phosphorylation is still clearly detectable after 6 days of incubation with TPA. These results on human rhabdomyosarcoma cells are, to our knowledge, the first evidence for a growth-inhibiting and a differentiative effect of TPA on a solid tumor of mesodermal origin.