Abstract Monoclonal antibodies against human prostatic acid phosphatase (PAPase) were produced by immunization of human primary spleen cell cultures. Dissociated spleen cells were cultured for 5–8 days in the presence of 100 ng/ml of PAPase and pokeweed mitogen (1:5000). Following immunization, B cells were isolated and infected with Epstein-Barr virus (EBV). Two weeks after EBV-transformation, cells were fused with either mouse myeloma cells (SP2/OAg14) or human/mouse heteromyeloma cells (SHM-D33). Hybrid clones were screened for anti-PAPase production. In 7 independent immunizations, the average fusion frequency was 3.6 per 10 6 lymphocytes. 18–32% of the hybridomas produced anti-PAPase; approximately 75% of these secreted IgM and 25% secreted IgG. Antibody specificity was determined by immunoassay and immunohistological studies. The procedures described here may be suitable for the production of human monoclonal antibody of a useful specificity.