Immunological therapy of BALB/c nude mice (nu/nu) implanted with human breast tumors, estrogen receptor negative MX-1 and estrogen receptor-positive MCF-7, was carried out with four monoclonal antibodies (MoAbs) raised against human milk fat globule membrane glycoproteins also present on normal breast epithelial cells. MoAbs injected singly or as a partial mixture arrested growth of the tumors but to a lesser extent than a mixture ("cocktail") of all four MoAbs. Two model systems were developed in order to examine the capabilities of the four MoAbs to arrest human mammary tumor growth. In the first model the ability of these MoAbs to arrest tumor growth during a 6- to 8-week period was tested by injection of the MoAbs immediately before and after implantation (passive immunization) and thereafter every other day. In the second model the effect of these MoAbs on established and growing tumors was tested. Using the cocktail in the passive immunization protocol, human mammary tumor growth in nu/nu mice was arrested either completely or averaging to one-tenth the size of the controls for those mice in which the tumors had taken. Other human carcinomas, colon and lung, under the same protocol, were not affected. Injection of cocktail every 2 days into nu/nu mice with established and growing human breast tumors (both estrogen receptor positive and negative) produced arrests of tumor growth of 44.1, 45.2, 49.8% of their controls after 7 to 8 days of treatment. Previously, it has been established that human mammary tumors are heterogeneous in expression of the human milk fat globule antigens recognized by our antibodies to the extent that some cells may have large amounts and others no detectable amount of a particular antigen. Those MX-1 tumors treated for a prolonged time with the cocktail of MoAbs that survived and continued to grow could be the result of the preferential multiplication of those cells in the heterogeneous population which had low or no antigen content. The breast tumors that did grow in the nu/nu mice after 8 weeks of injection of the cocktail revealed by immunoperoxidase staining a 90% reduction in the antigen content as recognized by these MoAbs when compared with untreated tumors. These results attest to the effectiveness of unconjugated anti-human milk fat globule MoAbs to arrest human breast tumor growth in nu/nu mice, and they also suggest that to best arrest tumor growth the use of a mixture of MoAbs should be considered.