Antisense DNA has shown an ability to target specific oncogene transcripts and inhibit their expression in cells, but the degree to which sustained treatment can suppress total levels of an oncogenic product and alter tumorigenesis in vivo remains to be determined. In this study, NIH-3T3 cells transformed by the activated c-Ha-ras oncogene from T24 human bladder cancer cells were treated for 3 consecutive days in vitro with an antisense DNA pentadecamer complementary to a target in the 5'-flanking region of the c-Ha-ras RNA transcript. Following antisense DNA treatment, a portion of the cells was lysed for measurement of RAS p21 while the remaining cells were evaluated for tumorigeneity by injection s.c. into athymic nude mice at a dose of 5 x 10(5) cells/mouse. The 3 days of treatment with the anti-c-Ha-ras DNA reduced RAS p21 cellular levels by more than 90% while a nonspecific control DNA reduced p21 levels by approximately 20%. Tumor growth of cells treated with anti-c-Ha-ras DNA was significantly reduced for up to 14 days following the end of treatment and implantation into the mice whereas the nonspecific control DNA had no significant effect. These effects on tumor growth were evident in two different strains of nude mice and in both males and females. It is suggested that the pronounced decrease in RAS p21 levels produced by anti-c-Ha-ras DNA resulted in a reversal of the transformed phenotype, and it is this reversal which accounts for the prolonged inhibition of tumorigenesis following antisense DNA treatment.