The glycolytic enzyme enolase (EC 4.2.1.11) exists as dimers formed from three structurally related subunits alpha, beta, and gamma, encoded by separate genes. The gene encoding the beta-subunit is expressed only in striated muscles. We have previously shown that the beta-enolase gene belongs to a small subset of muscle-specific genes showing transcriptional activity in cultured myoblasts, prior to withdrawal from the cell cycle. An increase in the level of beta-enolase mRNA occurs during terminal differentiation of myoblasts. To investigate the mechanisms underlying this increase, we have simultaneously estimated, under steady state conditions, the rate of synthesis and the stability of beta-enolase mRNA in proliferating C2.7 myoblasts as well as in differentiating myotubes. The method used is based on the isolation of newly synthesized RNA from the total RNA pool, following pulse-labeling of intact cells in the presence of 4-thiouridine. The results described here demonstrate a coordinate increase in newly synthesized and total beta-enolase mRNA, while the mRNA half-life, about 4 hr, remains unchanged in the course of terminal differentiation. The expression of the gene for insulin-like growth factor-II (IGF-II), a major positive regulator of myogenesis, was analyzed using the same approach. It is concluded that the up-regulation of beta-enolase as well as IGF-II gene expression in differentiating muscle cells reflects an increased rate of entry of newly synthesized mRNAs into the general pool of transcripts without changes in their respective half-lives.