Stable transfection of endothelial nitric oxide synthase (eNOS) cDNA in human oral carcinoma SCC-25 cells was performed. Two eNOS-expressing clones were isolated, and both were shown to have increased eNOS expression as assayed by Northern and Western analyses. Furthermore, a nitrite assay indicated that nitric oxide production in eNOS-transfected cells was increased. The growth rate and plating efficiency of eNOS-transfected cells in vitro were lower than that of the wild-type parental or vector control-transfected cells as assayed by growth curves, [3H]thymidine incorporation, and standard plating efficiency assays in L-arginine-rich medium. However, when these cancer cells were inoculated into nude mice, tumor size of eNOS-transfected cells was smaller during the first 25 days but increased later as compared to tumor size of parental and vector control-transfected cells. It is not clear whether the later increase in tumor size was due to an increase in SCC-25 cancer cell proliferation, normal stromal cell proliferation, or both. These results show significant effects of overexpression of eNOS on tumor growth in vitro and in vivo.