Radiation-induced bystander mutagenesis at a relatively low dose range was investigated using low LET beta-particles in a three-dimensional cell culture model. CHO cells were labeled with 0, 0.5, 1.0 or 5.0 muCi tritiated thymidine ((3)HdTTP) for 12 h and subsequently incubated with A(L) cells for 24 h at 11 degrees C. The cell mixture was centrifuged to produce a spheroid of 4 x 10(6) cells of which there was five times more A(L) than CHO cells. The short-range beta-particles emitted by (3)HdTTP result in self-irradiation of labeled CHO cells, thus biological effects on neighboring A(L) cells can be attributed to the bystander response. To evaluate such response, non-labeled bystander A(L) cells were isolated from among labeled CHO cells and studied independently for survival and mutagenesis. Treatment of CHO cells with (3)HdTTP resulted in a dose-dependent increase in bystander mutation incidence among neighboring A(L) cells compared to controls. In addition, multiplex PCR analysis revealed the types of mutants to be significantly different from those of spontaneous origin. These data provide evidence that low dose low LET radiation can induce bystander mutagenesis in a three-dimensional model. The results of this study will address the relevant issues of actual target size and radiation quality, and are likely to have a significant impact on our current understanding of radiation risk assessment.