The secretion of thymidine by mononuclear phagocytes was correlated with the activity of the enzyme thymidine kinase (TK). Macrophages cultured in regular tissue culture medium released thymidine and did not express TK. However, when macrophages were incubated with medium conditioned by L cells, they expressed TK, incorporated 3H thymidine into trichloroacetic acid precipitable material, and ceased to secrete the nucleotide. Furthermore, replicating P388/D1 cells were induced to secrete thymidine by inhibiting TK with d-glucosamine. These results have demonstrated an inverse relationship between thymidine secretion and the expression of TK. They suggest that thymidine secretion by macrophages may be attributed to their lack of TK activity.