Quiescent mouse Swiss 3T3 cells or human fibroblasts treated with interferon for only 2 hr before the addition of serum are effectively protected against virus infection for up to 24 hr after the addition of serum and the removal of interferon. In sharp contrast, exposure of quiescent fibroblasts to interferon only before addition of serum or the peptides epidermal growth factor and insulin, has no inhibitory effect on cell proliferation. Further, even when interferon is added after the addition of growth factors, an exposure of several hours is required to inhibit stimulation of DNA synthesis. These findings strongly suggest that the antiviral action of interferons is dissociable from their ability to inhibit cell proliferation. The implications of these findings for elucidating the molecular events leading to the biological responses elicited by interferon are discussed.