Stable clones of murine hybridomas 7E11-C5 and 9H10-A4 were obtained following immunization with LNCaP cells. The LNCaP cells were isolated from a human prostatic cancer (Ca). Both hybridomas secreted monoclonal antibodies (MoAb) of the IgG1 subclass which were reactive with the insoluble, cytoplasmic, membrane rich fractions of the immunogen. Neither MoAb reacted with the soluble cytosol of LNCaP cells nor with purified human prostatic acid phosphatase (PAP) nor prostate specific antigen (PSA). MoAb 9H10-A4 reactivity was very narrow and limited to the surfaces of LNCaP cells only. MoAb 7E11-C5 specificity was restricted to human prostatic epithelium, both normal and malignant. Except LNCaP, none of the 32 lines of human normal or neoplastic cells reacted with MoAb 7E11-C5. In a survey of frozen sections from 175 human specimens, positive indirect immunoperoxidase staining was limited to epithelium in all 11 specimens of localized and metastatic CaP, 7 benign prostatic hypertrophy (BPH) cases and 7 normal prostates. None of the 26 various nonprostatic tumors nor 120 out of 122 specimens from 28 different normal organs were reactive. Positive staining occurred in 2 out of 14 normal kidneys. Competitive binding with MoAb 7E11-C5 or its F(ab')2 fragments demonstrated the presence of circulating epitope 7E11-C5 in 20 out of 43 sera from CaP patients. Only 3 out of 66 sera from nonprostatic malignancies reacted. None of 30 normal blood donors sera nor 7 BPH sera were positive. Thus, highly significant (p less than 0.0001) association between diagnosed prostatic cancer and circulating molecules expressing the epitope reactive with MoAb 7E11-C5 was established. Significant probability (p less than 0.05) also suggested that patients with positive ELISA test are more likely to be in progression, than those who are negative. These results suggest that this apparently new antigenic marker may be of clinical potential in CaP.