We report the first mutational study of thymidine kinase 1 (TK1) performed in human solid tumors. We sequenced cDNAs representing the complete coding region of TK1 in human breast (n=22) and colorectal (n=26) cancer. Codon 106 near the ATP binding site constantly differed (ATG → GTG; Met → Val) from the one deposited by Bradshaw and Deininger in the Genbank database (Accession number NM_003258). Silent polymorphisms at codon 11 (CCC → CCT; Pro → Pro) and codon 75 (GCG → GCA; Ala → Ala) were frequently detected in tumors as well as in normal tissues. In breast cancer the two polymorphisms were observed in 63.6% of the samples analyzed. No significant association could be found between polymorphisms and TK activity. In colorectal cancer the incidence of the two changes was 73.1% and 69.2%, respectively. Interestingly, one colon cancer with high cytosolic TK activity displayed two missense mutations located in and near the putative phosphorylation site by tyrosine kinase (s) (TAT → CAT; Tyr → His) and by cAMP-, cGMP-dependent protein kinase (TAC → TGC; Tyr → Cys), respectively; adjacent normal mucosa showed no mutation. This may open new avenues that imply TK1 activity in tumor cell proliferation.