Serological analysis of a recombinant lung cancer cDNA expression library with the autologous patient serum led to the isolation of 20 clones representing 12 different genes: 4 of these were known genes, and the other 8 were previously unknown genes. Of the four known genes, aldolase A (NY-LU-1), previously shown to be overexpressed in lung cancer, was most frequently isolated. The other three genes were annexin XI, human HIV Rev-interacting protein Rip-1, and the human homologue of the ATP-binding arsA component of the bacterial arsenite transporter, all of which are known to be widely expressed in human tissues. Among the eight unknown genes, of most interest was NY-LU-12. Cloning of full-length NY-LU-12 showed that this cDNA was derived from the same gene as g16, a partially sequenced gene that mapped to the lung cancer tumor suppressor gene locus on chromosome 3p21. The reported g16 sequence, however, was significantly shorter (2433 versus 3591 bp). As a result of alternate splicing and subsequent frameshift, the reported g16 protein is 603 amino acids shorter than the NY-LU-12 product (1123 residues) at its COOH terminus and would therefore lack the epitopes recognized by the autologous serum. Analysis of the putative NY-LU-12 protein sequence predicted that it is a nuclear zinc finger protein with two RNA-binding domains, and Southern analysis showed that this gene is partially deleted in the lung cancer line NCI-H740 but not in nine other lung cancer lines. Screening of normal and cancer patient sera showed anti-NY-LU-12 seroreactivity in 2 of 21 allogeneic lung cancer patients but not in 24 patients with other tumors or in 16 sera from healthy donors. Comparison of NY-LU-12 cDNA from Lu15 tumor and normal lung tissue by DNA sequencing and/or single-strand conformation polymorphism analysis showed no evidence of mutation. Considering the high frequency of 3p21 alterations in lung cancer and the fact that the tumor suppressor gene or genes in this locus have not been identified, additional studies on the NY-LU-12 gene and its product are warranted.