We have estimated the rate of unscheduled DNA synthesis (UDS) in human lymphocytes from measurements of tritiated thymidine incorporation into double-stranded DNA (ds-DNA) during incubation of cells in vitro. Cells were not subjected to stresses except those associated with careful handling, or in certain experiments, mild heating or treatment with phytohaemagglutinin (PHA). Contribution of scheduled DNA synthesis (SDS) to incorporation was reduced by inhibiting replication and separating freshly replicated single-stranded DNA from repaired ds-DNA by chromatography. By increasing the incubation temperature, which decreases SDS and increases UDS, the residual contribution of scheduled DNA synthesis to thymidine incorporation into ds-DNA was estimated. Effects of increasing the number of cells in S-phase by phytohaemagglutinin were also investigated. Results suggest that: the rate of unscheduled DNA synthesis is about 500 +/- 100 thymidine molecules incorporated per cell per hour; a temperature-sensitive process, probably hydrolysis of DNA, contributes much of the damage repaired by UDS; background ionizing radiation contributes little to the damage; and damage caused by DNA hydrolysis is repaired much more efficiently than lethal damage caused by ionizing radiation. Large increases in incorporation into ds-DNA occurred when cells were stimulated with PHA.