Trichomonas vaginalis, a human protozoan parasite known to lack the capability of synthesizing purine and pyrimidine nucleotides de novo, was found also incapable of converting its ribonucleotides to deoxyribonucleotides. The only apparent means of providing deoxyribonucleotides for DNA synthesis relies on salvaging exogenous deoxyribonucleosides by a deoxyribonucleoside phosphotransferase activity in the T. vaginalis 10(5) g pelletable fraction. The activity, constituted by at least two isozymes I and II, can be solubilized by Triton X-100, has a pH optimum of 5.0-6.0, and recognizes only thymidine, deoxyadenosine, deoxyguanosine, and deoxycytidine as the phosphate acceptor. TMP, dAMP, dGMP, dCMP, dUMP, FdUMP, and p-nitrophenylphosphate can serve as phosphate donors. Enzyme I has been purified 10-fold by DEAE-Sepharose chromatography and Sephacryl 200 filtration, and is totally freed of the acid phosphatase of T. vaginalis. It has an estimated molecular weight of 200,000 and Km values of 2-3 mM for the four deoxyribonucleosides, which act on each other as competitive inhibitors. It also possesses phosphatase activity capable of hydrolyzing p-nitrophenylphosphate with a Michaelis constant of 0.74 mM. The rates of hydrolysis are enhanced by thymidine, which suggests that the latter may be the preferred phosphate acceptor, and Enzyme I may be, thus, more a transferase than a phosphatase. This enzyme could be a potential target for antitrichomonial chemotherapy.