Cytogenetic abnormalities and high-frequency allele losses involving the short arm of human chromosome 3 have been identified in a variety of histologically different neoplasms. These fmdings suggest that a tumorsuppressor gene or genes may be located in the region of 3pl4-p25, although there has been no definitive functional proof for the involvement of a particular region of 3p. We report a rapid genetic assay system that has allowed functional analysis of defined regions of 3p in the suppression of tumorigenicity in vivo. Interspecific microcell hybrids containing fragments of chromosome 3p were constructed and screened for tumorigenicity in athymic nude mice. Hybrid clones were obtained that showed a dramatic tumor suppression and contained a 2-megabase fragment ofhuman chromosomal material encompassing the region 3p2l near the interface with 3p22. With these hybrid clones, we have defined a genetic locus at 3p2l-p22 intimately involved in tumor suppression.