D98-OR is a tumorigenic subline of HeLa cells. We isolated nine subclones from D98-OR and examined their tumorigenicity in nude mice. Three, two, and four subclones were highly, weakly, and nontumorigenic, respectively. While they all contained two copies of intact chromosome 11, restriction fragment length polymorphism (RFLP) analysis revealed that the allelic composition of this chromosome differed among them. The highly tumorigenic subclones were heterozygous for the 11p and 11q loci, whereas those that were weakly or nontumorigenic were homozygous. Thus, the loss of one chromosome 11 with the duplication of another associated with the reduced tumorigenicity. Taken together with previous reports, our results indicate that a putative tumor suppressor gene on chromosome 11 controls tumorigenic expression in a gene dosage-dependent manner, and most importantly, suggested that the functional inactivation of the gene requires only a "one-hit" mutation.