Stimulation of normal human foreskin fibroblasts with platelet-derived growth factor (PDGF) was inhibited by the addition of the synthetic double-stranded RNA polyinosinic-polycytidylic acid (poly-I:C) as measured by incorporation of 3H-thymidine (3H-TdR). Single-stranded polycytidylic or polyinosinic acid had no effect. Double-stranded RNA is an inducer of interferon-beta (IFN-beta) in fibroblasts. On the mRNA level, an expression of IFN-beta 2 but not of IFN-beta 1 was seen after addition of PDGF and/or poly-I:C. The inhibition of PDGF-induced mitogenesis was completely blocked by an antiserum to IFN-beta. Poly-I:C did not interfere with PDGF binding to its receptor, nor did it block protein synthesis, indicating that the inhibition is not due to a nonspecific toxic effect of the double-stranded RNA but rather is mediated by IFN-beta. The present study implies that the IFN-beta system in fibroblasts is a very potent autocrine inhibitory pathway.