Addition of mouse interferon-alpha/beta (IFN) to confluent, quiescent BALB/c 3T3 (clone A31) mouse fibroblasts resulted in a block or delay in serum-induced activation of the cell cycle. It was necessary to add IFN within 6 hours after serum stimulation to inhibit nuclear labeling with [3H]thymidine. This is consistent with the time required for platelet-derived growth factor (PDGF) to induce cells to become competent to respond to additional growth factors present in platelet-poor plasma. Simultaneous addition of IFN with PDGF inhibited the PDGF-induced synthesis of a 29-kilodalton and a 35-kilodalton protein that normally occurs within 1 hour after PDGF addition. IFN also suppressed the general increase in protein synthesis that occurs by the fifth hour after PDGF addition. These results show that IFN antagonizes the action of PDGF, thereby interfering with the activation of Go cells for G1 traverse and S-phase entry.