Thymidylate synthetase (TS) is a key enzyme as a methyl donor in the methylation reaction from dUMP to dTMP. TS activity was assessed in various tissue of mammary disorders. The descending order of TS activity was as follows: cancer-positive nodes, primary cancers, cancer-negative nodes, benign lesions, and normal parenchyma. Significant differences in TS activity were found between the positive nodes and each of the other tissues (p < 0.01). In node-positive cases, a significant correlation in TS activity was found between the primary cancers and positive nodes (r = 0.616, p = 0.033). There was no correlation between the nodal status and the TS activity in primary cancers. In 11 of 12 cases, the TS activity of positive nodes was higher than the 'calculated' TS activity of the primary cancer, which was defined as the TS activity per unit weight of cancer cells. A significant correlation was found between the calculated TS activity and the mitotic frequency in primary cancers (r = 0.697, p = 0.0001). On the other hand, a significant correlation could not be found between the TS activity and the mitotic frequency in positive nodes (r = 0.364, p = 0.244).