Reactive oxygen species (ROS) are thought to promote muscle atrophy in chronic wasting diseases, but the underlying mechanism has not been determined. Here we show that H2O2 stimulates ubiquitin conjugation to muscle proteins through transcriptional regulation of the enzymes (E2 and E3 proteins) that conjugate ubiquitin to muscle proteins. Incubation of C2C12 myotubes with 100 microM H2O2 increased the rate of 125I-labeled ubiquitin conjugation to muscle proteins in whole cell extracts. This response required at least 4-h exposure to H2O2 and persisted for at least 24 h. Preincubating myotubes with cycloheximide or actinomycin D blocked H2O2 stimulation of ubiquitin-conjugating activity, suggesting that gene transcription is required. Northern blot analyses revealed that H2O2 upregulates expression of specific E3 and E2 proteins that are thought to regulate muscle catabolism, including atrogin1/MAFbx, MuRF1, and E214k. These results suggest that ROS stimulate protein catabolism in skeletal muscle by upregulating the ubiquitin conjugation system.