At a concentration of 0.5 to 3 mmol/l, ATP stimulates the activity of mitochondrial DNA polymerase of Neurospora crassa under the optimum reaction conditions; at higher concentrations, an inhibitory effect is observed. 4-Chloromercuribenzoate (1 mmol/L), a thiol inhibitor, decreases the enzyme activity two-fold, while N-ethylmalcimide (2 mmol/L) has no effect. Ethidium bromide (up to 10 mumol/L) and heparin (up to 0.4 micrograms/mL) reduce the activity by 60%. ddTTP does not affect the DNA polymerase reaction. The best in vitro template is the activated calf-thymus DNA.