To clarify the molecular structures of nonspecific cross-reacting antigens (NCAs), a family of glycoproteins antigenically related to carcinoembryonic antigen (CEA), in human granulocytes, we have screened a cDNA library of human leukocytes using a cDNA probe for the N-terminal domain (domain-N) of NCA-50, an NCA species in tumor cells. In 95 positive clones randomly selected, we identified six NCA or NCA-related cDNA clones including NCA-50, biliary glycoprotein protein a, and W272 (CGM6) which have previously been reported, and three new clones, W236, W264, and W282, encoding three novel NCA species. W236 and W264 consist of a domain-N, a putative transmembrane domain, and a possible cytoplasmic domain. The domain-N of W264 is 89% similar to that of NCA-50 at amino acid level, whereas the domain-N of W236 is only 49 and 43% similar to those of NCA-50 and pregnancy-specific beta 1-glycoprotein-11 (PSG11), respectively, indicating that W236 belongs to a new subfamily within the CEA family. The third clone W282 encodes a protein consisting of a domain-N virtually identical to that of W264 and a short hydrophilic C-terminal domain. W264 and W282 seem to be derived from a single gene by alternative splicing of RNA. These three new species are particularly unique in respect that they lack the repetitive immunoglobulin-related domains that have been universally found in the human CEA gene family members. The biochemical and immunochemical properties of the recombinant proteins of these cDNA clones, however, did not coincide with those of six NCA species previously identified in granulocytes at protein level, suggesting that, in granulocytes, there exist at least 12 NCA or NCA-related species whose expression is under complex control.