A low molecular mass (< 3 kDa) phagocytosis-inhibitory factor (PIF), was partially purified from human erythrocyte membranes. PIF inhibits latex phagocytosis and C, as well as FcR-mediated phagocytosis, by macrophage-like cells in a Ca(2+)-dependent manner. This phagocytosis-inhibitory activity is reversible because removal of PIF restores phagocytic capability of cells. After treatment with PIF, Mac-1 Ag (CR3 or CD11b) becomes almost undetectable on the cell surface by immunofluorescence staining using the mAbs D-12 and BEAR-1, whereas staining with the LM2/1 anti-Mac-1 mAb proved that Mac-1 is still present on the cell surface, thus, indicating a possible conformational change in Mac-1. PIF has no significant inhibitory effect on staining of CR1 (CD35), CR2, (CD21), 20-kDa homologous restriction factor (CD59), decay-accelerating factor (CD55), LFA-1 (CD11a), or p150.95 (CD11c). Although binding of Mac-1-bearing U-937 cells to C3bi-opsonized beads is completely blocked, binding via Con A and FcRs remains unaffected by PIF treatment. In addition to the suppressive effect on phagocytosis, inhibition of cell adhesion was observed as well. The inhibitory effect of PIF on cell adhesion is not monocyte specific, because after exposure to PIF the TGW neuroblastoma cell line lost its ability to attach to the tissue culture plate, but retained its ability for homotypic aggregation. The possibility that PIF is a natural regulator of erythrophagocytosis is suggested.