Normal mouse peritoneal macrophages maintained in a serum-free medium for 48-72 h and then stimulated with phorbol myristate acetate, zymosan or bacteria, released large amounts of hydrogen peroxide. Opsonized zymosan and bacteria stimulated greater release than their unopsonized counterparts. Enhanced peroxide production was not a consequence of increased uptake of particles. Addition of serum to the serum-free medium abolished activation. The addition of interferon-gamma to the serum-free medium enhanced the effect of the serum-free treatment of macrophages from C3H/HeJ mice but abolished the effect of serum free treatment of macrophages from CFLP mice. The results are discussed in terms of negative regulation of receptor-oxidase linkage by serum.