Abstract The levels of the four deoxyribonucleoside 5′-triphosphates have been measured during the cell cycle of selectively synchronised HeLa cells. All four pools show cyclic fluctuations. The pyrimidine deoxynucleotide pools increase sharply in size at about the same time as the onset of the S phase, while those of the purines increase slowly throughout the S phase. This difference in pattern may be correlated with the previously observed fluctuations during the cell cycle of thymidine and deoxycytidine kinases as against the constant levels of deoxyadenosine and deoxyguanosine kinases. It is concluded that pyrimidine deoxynucleotide biosynthesis is triggered at the same time as, or immediately prior to the initiation of DNA replication.