We have examined myosin heavy chain gene transcription in the mouse muscle cell line C2/7 under different culture conditions. Gene-specific probes for embryonic (MHCemb), perinatal (MHCpn), and adult (MHCIIB) MHC sequences were used in nuclear run-on experiments, and transcriptional levels compared with cytoplasmic RNA accumulation of the transcripts during muscle cell differentiation. Transcripts are not detectable in myoblasts. These three MHC genes are physically linked within 370 kb of each other. However, they are not activated coordinately, but show independent transcriptional regulation as muscle cells differentiate into myotubes and as myotubes mature in culture. Post-transcriptional mechanisms also regulate cytoplasmic RNA accumulation of these MHC genes.