This study asks whether nuclear and mitochondrial DNA replication are supplied by distinct and separately regulated precursor pools. Using improved methodology for extraction and quantitation of deoxyribonucleoside triphosphate pools from HeLa cells, we have confirmed and extended earlier findings of Bogenhagen and Clayton ((1976) J. Biol. Chem. 251, 2938-2944). The four mitochondrial dNTP pools actually expanded following treatment with antimetabolites, even while total cellular pools of dTTP and dGTP are being severely depleted. Ribonucleoside triphosphates also accumulate in mitochondria after antimetabolite treatment. This confirms the idea of distinct regulatory mechanisms affecting precursor supplies for nuclear and mitochondrial DNA. Mitochondrial dNTP pools are larger, in relation to the cellular complement of mitochondrial DNA than are the whole cell pools in relation to the chromosomal DNA complement. Also, of the four dNTPs, the most sensitive to antimetabolite depletion is dGTP. This indicates that dGTP depletion may be more significant than previously realized as an element of the cytotoxic effects of methotrexate and 5-fluorodeoxyuridine.