Phorbol esters selectively and reversibly disassemble the contractile apparatus of cultured skeletal muscle as well as inhibit the synthesis of many contractile proteins without inhibiting that of housekeeping proteins. We now demonstrate that phorbol esters reversibly decrease the mRNA levels of at least six myofibrillar genes: myosin heavy chain, myosin light chain 1/3, myosin light chain 2, cardiac and skeletal α-actin, and skeletal troponin T. The steady-state message levels decrease 50- to 100-fold after 48 h of exposure to phorbol esters. These decreases can be attributed at least in part to decreases in transcription rates. For at least two genes, cardiac and skeletal α-actin, some of the decreases are the result of increased mRNA turnover. In contrast, the cardiac troponin T steady-state message level does not change, and its transcription rate decreases only transiently upon exposure to phorbol esters. Phorbol esters do not decrease the expression of the housekeeping genes, α-tubulin, β-actin, and γ-actin. Phorbol esters do not decrease the steady-state message levels of MyoD1, a gene known to be important in the activation of many skeletal muscle-specific genes. Cycloheximide blocks the phorbol ester-induced decreases in transcription, message stability, and the resulting steady-state message level but does not block the tetradecanoyl phorbol acetate-induced rapid disassembly of the I-Z-I complexes. These results suggests a common mechanism for the regulation of many myofibrillar genes independent of MyoD1 mRNA levels, independent of housekeeping genes, but dependent on protein synthesis.