Co-administration of CpG-containing immunostimulatory oligodeoxynucleotides and parasite antigen protects susceptible BALB/c mice from otherwise progressive infection with Leishmania major. Although the protective effect of CpG-containing immunostimulatory oligodeoxynucleotides is clearly dependent on endogenous interleukin-12 and interferon-gamma production, the source of these Th1-promoting cytokines in infected mice is unknown. In contrast to macrophages from Leishmania-resistant C57BL/6 mice, macrophages from susceptible BALB/c mice are hyporesponsive to stimulation with lipopolysaccharide and interferon-gamma. While studying interactions of various antigen-presenting cells with Leishmania, we found that BALB/c inflammatory skin macrophages, whether Leishmania-infected or uninfected, produced large amounts of interleukin-12 when treated with CpG-containing immunostimulatory oligodeoxynucleotides. Like lipopolysaccharide, CpG-containing immunostimulatory oligodeoxynucleotides induced production of interferon-gamma and release of nitric oxide by skin macrophages. Studies using skin macrophages from interleukin-12- and interferon-gamma-deficient BALB/c mice demonstrated that nitric oxide release was not dependent on interleukin-12 and interferon-gamma production. Approximately 44% and 27% of intracellular Leishmania major amastigotes were killed by infected skin macrophages within 72 h upon stimulation with CpG-containing immunostimulatory oligodeoxynucleotides and lipopolysaccharide, respectively. Parasite killing by macrophages was independent of endogenous interferon-gamma production, but was strongly enhanced by exogenous interferon-gamma. Parasite elimination was dependent on the induction of nitric oxide, however. In vivo, injection of CpG-containing immunostimulatory oligodeoxynucleotides into lesional skin reduced the parasite burden approximately 50-fold within the first 5 d of infection prior to full generation of a Th response. These results suggest that skin macrophages, constituting the principal reservoir of parasites in infected susceptible mice, produce Th1-promoting cytokines in response to CpG-containing immunostimulatory oligodeoxynucleotides. In addition, CpG-containing immunostimulatory oligodeoxynucleotides may also act locally on skin macrophages to facilitate Leishmania clearance by inducing nitric oxide production.