The use of radiolabeled thymidine (TdR) and thymidine analogs as PET-based tracers of tumor growth rate is based on the assumption that measurement of uptake of these nucleosides, a function primarily of thymidine kinase-1 (TK₁) activity, provides an accurate measure of active cell proliferation in tumors. The goal of this study was to test this hypothesis and determine how well these tracers track changes in proliferation of tumor cells. TK₁ activity; S-phase fraction; and uptake of TdR, 3′-deoxy-3′-fluorothymidine (FLT), and 2′-fluoro-5-methyl-1-(β-D-2-arabino-furanosyl) uracil (FMAU) were determined in plateau-phase and exponentially growing cultures of 3 human and 3 murine tumor cell lines. TK₁ activity and S-phase fraction increased in all cell lines as cells moved from plateau-phase conditions to exponential growth. Some cell lines had relatively large TK₁ activities and S-phase fractions under plateau-phase conditions, consistent with a loss of normal cell cycle checkpoint control in these cells. There were also 2 cell lines in which TK₁ activity changed little as cells moved from the plateau phase to exponential growth, suggesting that in these cell lines, de novo nucleotide synthesis pathways predominate over salvage pathways. Both TdR and FLT detected changes in TK₁ activity. The slope of the relationship between TdR uptake and TK₁ activity was nearly twice that for FLT and more than 40-fold that for FMAU. Although not all tumors show a strong TK₁ dependence of proliferation, in all cell lines for which proliferation is highly TK₁ dependent, phosphorylation of TdR or FLT accurately reflects changes in TK₁ enzyme activity.