The effect of increasing temperature up to 43 degrees C on thymidine (TdR) salvage, i.e. uptake of TdR, thymidine kinase (TK) activity, intracellular amount of thymidinetriphosphate (dTTP) and specific activity of 3H-dTTP and incorporation of dTTP into DNA, was studied in ascites tumour cells in vitro. While the uptake of [3H]TdR was almost unchanged up to 40.5 degrees C and 2.4 h followed by a decrease at later times and higher temperatures, TK activity declined already after 30 min in a temperature-dependent way, leading to a decrease in cellular amount of dTTP reaching almost undetectable values at 43 degrees C and 6.0 h. The specific activity of [3H]dTTP in the cell was elevated at 39 degrees C and 43 degrees C, but somewhat reduced at 40.5 degrees C. Incorporation into DNA of [3H]TdR decreased in a temperature-dependent way with increasing incubation time. After correction for specific activity of [3H]dTTP, however, the incorporation of [3H]TdR into DNA at 39 degrees C and 40.5 degrees C was unchanged up to 2.4 h and for 40.5 degrees C up to 6.0 h, while incorporation into DNA at 43 degrees C was still reduced. The overall protein synthesis was also reduced in a temperature-dependent way. We concluded that phosphorylation of TdR to dTMP was more sensitive to moderately elevated temperatures than uptake of TdR and DNA synthesis per se, caused by a reduced TK activity. The decline in TK activity was probably due to a decrease in TK polypeptide synthesis.