The cDNA of mucin 4 (MUC 4) has been cloned from human tracheo-bronchial cDNA library and MUG 4 was demonstrated to be expressed in normal tracheobronchial and colonic mucosa. However, as there are no antibodies available, there is little information on the distribution of MUC 4 on normal and malignant tissues. A 22 amino-acid peptide (M4.22), derived from the MUC 4 cDNA sequence was synthesized and used to immunise mice and 2 monoclonal antibodies were produced and characterized by ELISA, immunoperoxidase staining and flow cytometry. By ELISA the two anti-MUG 4 mAbs reacted with the immunizing peptide (M4.22), but not with peptides derived from other mucins. By immunoperoxidase staining the mAbs reacted with native mucin expressed in normal colon and to a lesser extent with stomach, but not with normal lung. By contrast, the mAbs reacted most strongly (similar to 70%) with lung cancers. Using six-mer overlapping peptides the minimum number of amino acids in the epitopes detected by the mAbs were TPL (M4.171) and PLPV (M4.275). MUC 4 detected by the mAbs was of a molecular mass (180 kDa) by Western blotting. Using anti-MUC 4 mAbs non-large cell lung cancers were found to be MUC 4 positive compared with normal lung tissues which were negative. By contrast, normal colon was strongly positive but the expression was decreased in colon cancer. These findings could have diagnostic and therapeutic implication for lung cancer.