This study was designed to gain an insight into mechanisms by which cytochrome c gene expression is enhanced by increased contractile activity in skeletal muscle. When rat tibialis anterior muscles were stimulated (10 Hz, 0.25 ms) for 0, 2, 6, 12, or 24 h or 2, 5, 9, or 13 days (n = 4 for each time point), cytochrome c protein (enzyme-linked immunosorbent assay) and mRNA (Northern blot analysis) concentrations started to increase by 9 days, and this was associated with concurrent decreases in cytochrome c mRNA-protein interaction (RNA gel mobility shift assay). We found that the decreased RNA-protein interaction in the stimulated muscle extract was restored by ultracentrifugation (150,000 g, 1 h) in the supernatant fraction. The 150,000 g pellet fraction of stimulated muscle was capable of inhibiting the RNA-protein interaction in control tibialis anterior muscles. These results provide evidence of an inhibitory factor that is responsible for decreasing RNA-protein interaction in the 3'-untranslated region of cytochrome c mRNA in continuously stimulated muscle.