Despite abundant molecular and cytogenetic evidence supporting the role of tumor suppressor genes (TSGs) in nasopharyngeal carcinoma (NPC), the specific molecular genetic basis for this cancer requires further elucidation. A functional approach utilizing monochromosome transfers of intact and truncated chromosomes was used to evaluate the ability of chromosomal regions to functionally suppress tumor growth in a nude mouse system. Resultant hybrids were analyzed by microsatellite and fluorescent in situ hybridization (FISH) techniques to confirm the success of transfer. Thus, microcell hybrids were established for chromosomes 3, 9, and 11. Tumor suppressive regions were localized to 3p21.3, 9p21, 11q13, and 11q22-23 by analyzing deletion panels of microcell hybrids for chromosome 3, by transfection studies for chromosome 9, and by analysis of tumor segregants for chromosome 11. It is clear from these functional studies that multiple tumor suppressive regions are involved in NPC development. Localizing the genes to specific chromosomal regions using this functional approach is expected to greatly facilitate the eventual identification of TSGs essential for NPC development.