The activity of deoxycytidine kinase (EC 2.7.1.74), an important pyrimidine salvage enzyme, was elevated 5- to 30-fold in human ovarian carcinoma and OVCAR-5 cells, in human colon carcinoma and HT-29 cells, in rat hepatoma 3924A solid tumors and cells, and in rat sarcoma as compared with the respective control normal cells. There was an inverse relationship between cell doubling time and deoxycytidine kinase activity in 8 cancer cell lines, with rapidly growing HL-60 cells (20 hr) showing the highest, and slower-growing lung H69 cells (60 hr) the smallest, increase in enzyme activity. In time-sequence studies in human HL-60, OVCAR-5, PANC-1, and rat hepatoma 3924A cells, there was a significant rise in deoxycytidine kinase activity after 3-6 hr of seeding, with peak increases (3.5- to 4-fold) at 48-72 hr in the log phase in comparison with values of the respective plateau phase cells (96-144 hr). In extracts of various cancer cells, the high deoxycytidine kinase activity was competitively inhibited by difluorodeoxycytidine (DFDC), with Ki = 7 to 30 microM. The Km for deoxycytidine in various carcinoma cell lines ranged from 0.3 to 0.7 mM and addition of DFDC increased the apparent Km from 0.7 to 4 mM. Deoxycytidine kinase activity in human HL-60 cells was inhibited by the end product, dCTP, with IC50 = 3 microM; dCTP elevated the Km for deoxycytidine from 0.35 to 0.9 mM. dTTP reversed the inhibition by dCTP.(ABSTRACT TRUNCATED AT 250 WORDS)