The adjuvant muramyl dipeptide (MDP) has been shown to affect a number of macrophage functions in vitro. We studied the effect of subcutaneous injection of MDP into mice. Cultured peritoneal macrophages from treated mice displayed increased spreading, total cell protein, and specific activity of beta-glucosaminidase a constituent of macrophage lysosomes, and of lactate dehydrogenase. Generation of superoxide anion (O2-) by MDP-treated macrophages stimulated by contact with phorbol myristate acetate was enhanced by over fivefold to levels achieved by macrophages from bacillus Calmette-Guérin-infected mice. The enhancement in stimulated O2- release was noted by 1 h after injection of MDP, peaked by 3 h, and remained high for at least 48 h. Priming for enhancement of O2- release by MDP was similar in athymic nude mice and in normal littermates, suggesting that mature T lymphocytes are not involved in this MDP effect. Priming for enhanced stimulated O2- release, and morphologic and enzymic changes, were not achieved by injection of the D-D stereoisomer of MDP. Phagocytosis of Candida albicans was only slightly greater by macrophages from mice give MDP, but MDP-stimulated cells killed two times more C. albicans in vitro than did cells from untreated animals. When MDP was given 18 h before, simultaneously with, or 24 h after lethal infectious challenge with C. albicans, treated mice were protected compared with controls. These results suggest that injection of MDP effectively and rapidly activates macrophages in the recipient animal. This agent should serve as an important probe of macrophage physiology and, perhaps ultimately, as a means of enhancing host defense in humans.