The LK26 antigen is a cell surface glycoprotein (M(r)35,000 to 40,000) of normal placenta and gestational choriocarcinomas that shows highly restricted distribution in normal tissues, being expressed primarily in a subset of simple epithelia. In this study, immunohistochemical methods were used to examine LK26 expression in 78 ovarian tumors and > 400 tumors of other histological types. Ovarian carcinomas derived from coelomic epithelium showed the most consistent and strongest immunostaining for LK26, with 52 of 56 cases being LK26+. Ovarian tumors of sex cord, germ cell, and stromal origin were generally LK26-. LK26 was not found in normal fetal or adult ovary; however, it was present in the lining epithelia of some benign ovarian cysts. Mesotheliomas, which share a common mesothelial origin with LK26+ ovarian tumors, expressed no or only low levels of LK26. Other epithelial cancers expressed LK26 in subsets of cases and generally showed heterogeneous or weak immunostaining; this group of LK26+ tumors includes endometrial (10 of 11 cases tested), colorectal (six of 27), breast (11 of 53), lung (six of 18), and renal cell (nine of 18) carcinomas. Four of five brain metastases derived from epithelial cancers and three of 21 neuroendocrine carcinomas showed prominent LK26 immunoreactivity. Only rare neuroectodermal tumors (two of 70) and none of the sarcomas (none of 58) or lymphomas tested (none of 21) were LK26+. Tests with cultured cells showed that the LK26 proteins expressed in choriocarcinoma and ovarian cancer cells are biochemically similar, and transfection experiments identified LK26 as an adult-type, high-affinity folate-binding protein. The present study provides the first detailed specificity and sensitivity analysis for folate-binding protein/LK26 in human tumors and defines a role for folate-binding protein/LK26 in immunobiological studies of ovarian cancers and other LK26+ neoplasms.