Although acquired immunity to leishmaniasis is generally considered to be cell mediated, humoral factors may be partially responsible. The present study showed that antisera from C57BL/6J mice superinfected with Leishmania donovani contained cytophilic antibody and opsonins for both the amastigote and promastigote stages of the parasite. Macrophages treated with mouse hyperimmune serum in an in vitro macrophage culture system bound statistically significantly more parasites at 4 degrees C (and subsequently phagocytized them at 37 degrees C) than did macrophage cultures treated with control serum. The percentages of antibody-treated macrophages bearing and containing parasites were also significantly greater than the percentages of control serum-treated macrophages bearing and containing parasites, respectively. These differences persisted in cultures during a 9-day observation period when sera from mice killed 10 or 11 days after superinfection were used. However, when sera from mice killed 24 days after superinfection were tested with amastigotes, by day 9 the number of parasites and the percentage of cells parasitized in the culture decreased to control values or significantly below them. Thioglycolate-stimulated macrophages treated with hyperimmune serum bound more amastigotes at 4 degrees C than did stimulated macrophages treated with control serum. Activated macrophages also demonstrated increased nonspecific binding of amastigotes. Treatment of macrophages with trypsin reduced both cytophilic antibody-specific and nonspecific binding of amastigotes. The demonstration of in vitro effects of anti-leishmanial antibody from superinfected mice might indicate a possible role for humoral antibody in immunity to leishmaniasis in mice.