We previously have reported the presence of interferon-beta 2 (IFN-beta 2) mRNA in PHA-stimulated human peripheral blood leukocytes (PBL), as well as in nonstimulated cells, although at a lower level. The IFN-beta 2 cloned from a leukocyte library appeared to be similar to that of the fibroblast IFN-beta 2 gene first described in fibroblasts. To assess the nature of the cell population in which the synthesis of IFN-beta 2 takes place, PBL were fractionated in adherent and nonadherent cells. The antiviral activity of the culture supernatants of adherent cells was characterized as the IFN-beta type by neutralization with polyclonal antibodies raised against purified fibroblast IFN-beta 2. IFN-beta 2 mRNA was observed in enriched monocyte populations and accumulated very rapidly, peaking at 2.5 h. RNA extracted from these cultures encoded in a reticulocyte lysate a protein immunoprecipitated by the anti-IFN-beta 2 antiserum. In addition, IFN-beta 2 secreted in monocyte supernatants also was immunoprecipitated by the specific antiserum and was able to compete with the fibroblast IFN-beta 2, suggesting a strong similarity between the fibroblast and monocyte proteins.