The rat myoblast L 6E 9 cell line under appropriate culture conditions is a uniform population of cycling cells which can be induced to differentiate into a pure population of myotubes. The pattern and kinetics of myogenic differentiation of this cell line are similar to those of primary skeletal muscle myoblasts. We have used this cell line to investigate the controls regulating the synthesis and accumulation of myosin heavy chain during myogenic development. From pulse labeling studies of total cellular protein synthesis, we observed that activation of MHC § § Abbreviations used: MHC, myosin heavy chain. synthesis is temporally correlated with cell fusion and myotube formation. MHC synthesis is transiently induced from <1% up to 25% of the total protein synthesized. After MHC has accumulated to the steady-state level characteristic of fully differentiated myotubes, MHC synthesis decreases very rapidly to almost basal levels. To determine whether this transient induction of MHC synthesis was due to parallel changes in MHC messenger RNA levels, the accumulation and compartmentalization of MHC mRNA during L 6E 9 cell differentiation was followed by complementary DNA/RNA hybridization using cDNA prepared against MHC mRNA purified from L 6E 9 cells. We demonstrate that the level of MHC synthesis closely parallels the level of cytoplasmic MHC mRNA. The induction of MHC mRNA accumulation is initiated at least 36 hours prior to cell fusion and at a time when all cells in the population are still uncommitted to terminal differentiation as tested by cell cloning. The level of cytoplasmic MHC mRNA is increased from ~200 molecules per cell in the growing state to ~50,000 molecules at the peak of induction (day 6 after plating). Subsequently the levels of MHC mRNA decrease very rapidly and at day 10 after plating there are only ~3000 molecules per myotube nucleus. A striking feature of this regulation is the behavior of MHC mRNA on oligo(dT) columns. Most (~90%) of the MHC mRNA transiently induced during differentiation has a very short poly(A) tail (<20 nucleotides). We conclude that the striking induction followed by deinduction of MHC synthesis is controlled primarily by the induction and deinduction of cytoplasmic MHC mRNA accumulation. The relationship of our observations to muscle physiology is discussed.