High frequencies of allelic loss on the short arm of chromosome 3 in small cell lung cancer (SCLC) and a number of other tumors suggest the existence of a tumor suppressor gene(s) within the deleted regions. Two small cell lung cancer lines, NCI H740 and GLC20, have been described which have homozygous deletions in the region 3p21.3. The deleted region overlaps with a 2 Mb fragment of human DNA present in the interspecies hybrid HA(3)BB9F, that suppresses tumor formation by mouse A9 fibrosarcoma cells. Human sequences from this cell hybrid were isolated using inter Alu PCR. From this starting point, a P1 contig was developed for the region of 450 Kb that is common to the homozygous deletions seen in the SCLC lines NCI H740 and GLC20 and is also present in HA(3)BB9F, the suppressed A9 hybrid. Individual P1 clones were assayed for their ability to suppress the tumorigenicity of the mouse fibrosarcoma cell line A9 as assayed by injection of transfected A9 cells into athymic nude mice. The introduction of one of the P1 clones into A9 cells resulted in suppression of tumor growth whereas two other P1 clones from the contig failed to suppress tumor formation in athymic nude mice. These data functionally delimit a tumor suppressor locus to a region of 80 kb within a P1 clone at 3p21.3.