The WWOX gene encodes a tumor suppressor spanning the second most common human fragile site, FRA16D. Targeted deletion of the Wwox gene in mice led to an increased incidence of spontaneous and ethyl nitrosourea-induced tumors. In humans, loss of heterozygosity and reduced or loss of WWOX expression has been reported in esophageal squamous cell cancers (SCC). In the present study, we examined whether inactivation of the Wwox gene might lead to enhanced esophageal/forestomach tumorigenesis induced by N-nitrosomethylbenzylamine. Wwox+/- and Wwox+/+ mice were treated with six intragastric doses of N-nitrosomethylbenzylamine and observed for 15 subsequent weeks. Ninety-six percent (25 of 26) of Wwox+/- mice versus 29% (10 of 34) of Wwox+/+ mice developed forestomach tumors (P = 1.3 × 10-7). The number of tumors per forestomach was significantly greater in Wwox+/- than in Wwox+/+ mice (3.2 ± 0.34 versus 0.47 ± 0.17; P < 0.0001). In addition, 27% of Wwox+/- mice had invasive SCC in the forestomach, as compared with 0% of wild-type controls (P = 0.002). Intriguingly, forestomachs from Wwox+/- mice displayed moderately strong Wwox protein staining in the near-normal epithelium, but weak and diffuse staining in SCC in the same tissue section, a result suggesting that Wwox was haploinsufficient for the initiation of tumor development. Our findings provide the first in vivo evidence of the tumor suppressor function of WWOX in forestomach/ esophageal carcinogenesis and suggest that inactivation of one allele of WWOX accelerates the predisposition of normal cells to malignant transformation.