Monoclonal antibodies were raised against the detergent-insoluble cytoskeletal fraction of the human bladder carcinoma cell line, EJ. By immunofluorescence, one of these antibodies, H10-1, localized to keratin filaments in EJ and three out of five other transitional-cell carcinoma lines. Primary normal-human urothelial cells in culture were not recognized by H10-1. Strong staining of keratin filaments was also seen in 11 human adenocarcinoma cell lines, but it was only seen in a subpopulation of cells in one out of four squamous-cell carcinoma lines and not at all in two normal diploid human epidermal keratinocyte strains. Immunofluorescence on frozen sections of mouse, human, and rabbit tissues showed that H10-1 recognized the upper layer of the transitional epithelium of mouse and rabbit bladder, and a subset of simple epithelia (mouse stomach glands and some colon mucosal glands, but not kidney tubules, small intestine, ovary germinal epithelium, or endometrium; some human colon mucosal glands and glandular breast epithelium, but not endometrium). The antigen which is recognized by this antibody was not detected in examples of stratified squamous epithelium (mouse skin, nonglandular stomach, or esophagus; human skin, esophagus, or rectum) or nonepithelial tissue. On frozen sections of human tumors, the H10-1 antibody recognized 9 out of 14 colon carcinomas and 4 out of 6 breast carcinomas but did not recognize two sarcomas or a melanoma. This antibody apparently does not recognize its antigen in the presence of sodium dodecyl sulfate (SDS) because brief exposure of methanol-fixed cells to 0.1% SDS reversibly inhibited the binding of the H10-1 monoclonal antibody to keratin filaments by immunofluorescence, even when the binding of a rabbit polyclonal antikeratin antibody was unaffected. Two-dimensional gel-electrophoretic analysis of keratin-enriched fractions which had been isolated from various cell lines disclosed a good correlation between the presence of a 47-kdalton, pI-5.35 polypeptide and positive immunofluorescence with this antibody. These observations suggest that a certain keratin or keratin-associated protein is specifically expressed in a subpopulation of transitional and simple epithelial cells, and cultured cells derived from these epithelia. The monoclonal antibody described here may therefore be useful for increasing the precision of histological classification of epithelia, as well as the verification of the origin of certain cultured cell lines.