In order to test the validity of using 3H-uridine-induced chromosome damage as a measure of the transcriptional activity of chromosomes, experiments were performed to examine the mechanism by which 3H-uridine induces sister chromatid exchanges. DON and B14FAF28 Chinese hamster cells exposed to 3H-uridine showed a dose-dependent increase in SCE frequency, whereas unlabelled uridine produced no increase. 3H-uridine labelling in the presence of increasing concentrations of unlabelled uridine eliminated this increase in a concentration dependent manner. Competition between 3H-uridine and other unlabelled pyrimidines (thymidine and cytidine) was equally as effective; however, unlabelled hypoxanthine had no effect on 3H-uridine-induced SCEs. This suggested that 3H-uridine caused SCEs after interconversion into deoxycytidine followed by incorporation into DNA. Significant incorporation of 3H into DNA was confirmed by direct comparisons of label in the DNA and RNA fractions, and about 90% of this label was found to be in the cytosine fraction of hydrolysates. We therefore conclude that 3H-uridine produces chromosome damage because of the incorporation of a tritiated conversion product into DNA, rather than by exposure of transcriptionally active DNA to 3H-uridine-labelled RNA. We suggest therefore, that the results of earlier experiments, in which 3H-uridine chromosome damage has been used to assay the transcriptional activity of chromosomes, may need to be reinterpreted.