Gelatin microspheres containing recombinant human interferon αA/D (A/D‐IFN) (IFN‐microspheres) potentiated the antitumor activity of mouse peritoneal macrophages (Mφo) much more efficiently than free A/D‐IFN. Mφo acquired the inhibitory activity on tumor cell growth by the ingestion of IFN‐microspheres without the aid of lipopolysaccharide (LPS), though LPS was required as a second signal for activating Mφo primed with free IFN. The IFN‐microspheres were much more efficient than free IFN plus LFS in respect of the IFN amount and the time required for Mφo activation. Furthermore, Mφo pretreated with the IFN‐microspheres maintained their activated state for a much longer period than those pretreated with free A/D‐IFN plus LPS. A monoclonal anti‐IFN‐αA antibody, which was capable of neutralizing A/D‐IFN, did not interfere with the Mφo activation by the IFN‐microspheres. Even human IFN‐αA was effective in activating murine Mφo similarly to A/D‐IFN, when given in the form of IFN‐microspheres, though human IFN‐αA in the free form was ineffective. These results argue that the mechanism of Mφo activation by the IFN‐microspheres is different from that by free IFN.