Macrophages were identified to be a major source of interferon produced in murine spleen cell cultures after intravenous injection of Corynebacterium parvum (C. parvum), strain CN 6134 or Bacille Calmette Guérin (BCG). Another strain of C. parvum, CN 5888, which lacks RES stimulating activity and adjuvant activity in vivo, was not effective when injected intravenously. Protein synthesis was required for interferon activity to be produced and protein synthesis was also required for the antiviral state to be expressed. The antiviral activity was relatively stable to pH 2 and neutralized by an antiserum against virus-induced fibroblast interferon, thus exhibiting some properties of type I interferon. In vitro only CN 6134, the biologically active strain, could induce small amounts of interferon in spleen macrophage cultures. Macrophages from CN 6134 or BCG-infected athymic nu/nu mice produced similar interferon titers as their controls. It is concluded that infection with certain immunomodulators can activate splenic macrophages via a predominantly T-cell independent mechanism. Interferon in turn may operate locally as a mediator of immunoregulation.