Nucleoside analogues are widely used as chemotherapeutic agents in the treatment of cancer. Several cancers are reported to be associated with mycoplasmas (i.e. Mycoplasma hyorhinis), which contain a number of nucleoside-metabolizing enzymes. Pyrimidine nucleoside analogues, such as 5-fluoro-2'-deoxyuridine (FdUrd), 5-trifluorothymidine (TFT) and 5-halogenated 2'-deoxyuridines can be degraded by thymidine phosphorylase (TP) to their inactive bases. We found in M. hyorhinis-infected MCF-7 breast carcinoma cells (MCF-7/HYOR) a mycoplasma-encoded TP that dramatically (20-150-fold) reduces the cytostatic activity of these compounds. The reduction in cytostatic activity could be fully restored in the presence of TPI (5-chloro-6-[1-(2-iminopyrrolidinyl)methyl]uracil hydrochloride), a known inhibitor of human TP. This observation is in agreement with the markedly decreased formation of active metabolite (i.e. FdUMP for FdUrd) or diminished drug incorporation into nucleic acids (i.e. for TFT and 5-bromo-2'-deoxyuridine) in MCF-7/HYOR cells compared with uninfected MCF-7 cells. Antimetabolite formation is fully restored in the presence of TPI. In contrast, 5-fluoro-5'-deoxyuridine (5'DFUR), an intermediate metabolite of capecitabine, was markedly more cytostatic in MCF-7/HYOR cells than in uninfected cells, due to the activation of this prodrug by the mycoplasma-encoded TP. Thus, our data reveal that M. hyorhinis expresses a TP that activates 5'DFUR but inactivates FdUrd, TFT and 5-halogenated 2'-deoxyuridines, and that is highly sensitive to the inhibitory effect of the TP inhibitor TPI. Given the association of M. hyorhinis with several human cancers, our findings suggest that pyrimidine nucleoside-based but not 5FU-based anti-cancer therapy might be more effective when combined with a mycoplasmal TP inhibitor.