Mixed allogeneic chimeras are emerging as a prospective approach to induce immune tolerance in clinics. However, the immunological function of macrophages in mixed chimeras has not been evaluated. Using a B6-->BALB/c mixed chimera model, we investigated the phenotype and function of F4/80(+) resident peritoneal exudate macrophage (PEMs) and splenic macrophages (SPMs) in vitro and in vivo. Recipient F4/80(+)PEMs and SPMs in mixed chimeras expressed significantly lower levels of MHC-II, CD54, and CD23 than those in non-chimeric mice before lipopolysaccharide stimulation. Recipient F4/80(+)PEMs and SPMs in mixed chimeras induced normal cell proliferation and delayed-type hypersensitivity of allo-T cells, but they induced more IFN-gamma and IL-2 products and less IL-10 and TGF-beta products of allo-T cells compared with those of non-chimeras. Furthermore, recipient F4/80(+)PEMs and SPMs had significantly higher phagocytotic capacity against chicken red blood cells or allo-T cells than those of controls while they had normal phagocytosis to Escherichia coli. Although some slight but significant alterations of recipient macrophages have been detected, these results provide direct evidences for the efficient immunity of recipient macrophages in mixed allogeneic chimeras. The present study also, for the first time, offered basic information for macrophages maturing in heterogeneous environments.