A monoclonal antibody (MAb) was generated by immunizing BALB/c mice with homogenized breast cancerous tissues. This antibody (PR81) was found to be of IgG(1) class and subclass, containing kappa light chain. PR81 reacted with either the membrane extracts of several breast cancerous tissues or the cell surface of some MUC1 positive cell lines (MCF-7, BT-20 and T-47D) tested by enzyme immunoassay and for MCF-7 by immunofluorescence method. PR81 also reacted with two synthetic 27 and 16-amino acid peptides, TSA-P1-24 and A-P1-15, respectively, which included the core tandem repeat sequence of MUC1. However, this antibody did not react with a synthetic 14 amino acid peptide that has no similarity with tandem repeat found in MUC1. The generated antibody had good and similar affinities (2.19 x 10(8) M(-1)) toward TSA-P1-24 and A-P1-15, which are mainly shared in the hydrophilic sequence of PDTRPAP. Through Western blot analysis of homogenized breast tissues, PR81 recognized only a major band of 250 kDa. This band is stronger in malignant tissue than benign and normal tissues.