Thiol antioxidants, including N-acetyl-L-cysteine (NAC), are widely used as modulators of the intracellular redox state. We investigated the hypothesis that NAC-induced reactive oxygen species (ROS) signaling perturbs cellular proliferation by regulating the cell cycle regulatory protein cyclin D1 and the ROS scavenging enzyme Mn-superoxide dismutase (MnSOD). When cultured in media containing NAC, mouse fibroblasts showed G(1) arrest with decreased cyclin D1 protein levels. The absence of a NAC-induced G(1) arrest in fibroblasts overexpressing cyclin D1 (or a nondegradable mutant of cyclin D1-T286A) indicates that cyclin D1 regulates this G(1) arrest. A delayed response to NAC exposure was an increase in both MnSOD protein and activity. NAC-induced G(1) arrest is exacerbated in MnSOD heterozygous fibroblasts. Results from electron spin resonance spectroscopy and flow cytometry measurements of dihydroethidine fluorescence showed an approximately 2-fold to 3-fold increase in the steady-state levels of superoxide (O(2)(*-)) in NAC-treated cells compared with control. Scavenging of O(2)(*-) with Tiron reversed the NAC-induced G(1) arrest. These results show that an O(2)(*-) signaling pathway regulates NAC-induced G(1) arrest by decreasing cyclin D1 protein levels and increasing MnSOD activity.