MAb KI recognizes a cell-surface glycoprotein (MW approximately 40 kDa) present in ovarian carcinomas, malignant mesotheliomas, squamous-cell carcinomas and normal mesothelial cells. In this study, expression screening was used to isolate cDNA clones encoding an antigen recognized by MAb KI from a cDNA library made from a human ovarian carcinoma cell line (OVCAR-3). Subsequently, other clones were isolated by DNA hybridization using a cDNA probe derived from one of the initial clones. The sequence of all the clones was similar. The longest cDNA contains 2,444 base pairs, and encodes a polypeptide of 263 amino acids with a calculated molecular weight of 30,511 daltons. The nucleotide sequence and deduced amino-acid sequence of the protein show no homology to other sequences in current data bases. In vitro translation of RNA transcripts from the cDNA inserts yielded polypeptides of 29 and 30 kDa. Similar-sized proteins were obtained upon expression of the cDNA in Escherichia coli, and these proteins were reactive with MAb KI. The protein(s) expressed in E. coli were purified and used to make rabbit or mouse antisera. These antisera reacted strongly with a soluble cytosolic protein in OVCAR-3 cells, but not with the membrane-bound antigen. Soluble cytosolic proteins of a similar size, recognized with MAb KI, were found in OVCAR-3 and N87 (gastric cancer) cells but not in 10 other cancer cell lines. These data indicate that the cloned cDNA encodes a cytosolic protein that reacted with MAb KI. This soluble protein is expressed only in cells containing the CAKI surface glycoprotein, suggesting that the 2 proteins could be structurally related.