The 12.9-kb human thymidine kinase gene (tk) has been sequenced in its entirety along with flanking regions. Consistent with the previously sequenced chicken tk sequence, the human tk is composed of seven exons. The intron sizes differ substantially, and are responsible for the four-fold greater size of the human relative to the chicken gene. Within the introns are found 13 Alu family repeated sequences and a polypyrimidine stretch. A functional promoter region has been located by fusing sequences from the 5' end of the tk gene to the chloramphenicl acetyl transferase (CAT) gene and assaying CAT activity following transfection into mouse L cells. Several putative transcription signals have been identified in the 5' end including 'TATAA' and 'CCAAT' sequences and 'G-C' elements, two of which are arranged in a 27-bp inverted repeat. There is also a 12-bp repeat, containing an inverted 'CCAAT' element. This repeat shows strong homology to a repeat in the chicken tk promoter as well as the 5' regions of other cell-cycle regulated genes, suggesting that it may be part of the promoter or a regulatory signal. The 5' flanking sequence is G + C-rich and has a high concentration of CpG dinucleotides.