When quiescent cells are stimulated to enter the cell cycle, the thymidine kinase (TK) gene is transcriptionally activated at the border of G1 and S. In this report we show that the human TK promoter contains multiple protein-binding sites. By site-directed mutagenesis, we identified a protein-binding site on the human TK promoter required for conferring G1-S-regulated transcription to a heterologous promoter and dissociated it functionally from an adjacent protein-binding domain containing an inverted CCAAT motif required for high basal level expression. Substitution-mutation of this site results in constitutive expression of the neo reporter gene in serum-stimulated fibroblasts, as well as in cells arrested in mid-G1 by a temperature-sensitive mutation. The regulatory domains for the human TK promoter exhibit interesting symmetrical features, including a set of CCAAT motifs and sites similar to the novel Yi protein-binding site recently discovered in the mouse TK promoter.