While loss of the Y chromosome from the karyotype of tumor cells has frequently been found in a number of human malignancies of different types, structural alterations are a much less common finding. Prompted by the high frequency of cytogenetic Y chromosome loss found in primary non-small-cell lung cancer (NSCLC), and the fact that NSCLC karyotypes usually contain marker chromosomes of unidentified origin, we have determined the Y chromosome status of 12 NSCLC samples (7 cell lines and 5 primary tumors) at a molecular level. Of the 9 cases which did not have a cytogenetically detectable Y chromosome, 4 were negative for all the Y sequences tested. The other 5, in contrast, retained some Y chromosome sequences. In 1 case (H520), only Yq heterochromatic sequences were detected, whereas in the remaining 4 (L162, L93, L125 and L71) both Yq heterochromatic sequences and Y euchromatic sequences were retained. The region of common overlap for loss of Y euchromatin was Yp distal to the Y centromere. We hypothesize that deletion of Yp sequences may play a role in tumor progression in NSCLC due to loss of a tumor-suppressor gene.