A microbial fermentation process could be used to produce thymidine biologically but many of the enzymes related to nucleotide biosynthesis are highly regulated. To overcome the complex regulation steps, an analogue mutant of Brevibacterium helvolum resistant to fluorouracil, hydroxyurea, and trimethoprim was constructed. This mutant accumulated 380 mg thymidine l(-1) in 16 h in shake-flask culture. However, the accumulation of thymidine monophosphate (TMP) inside the cells suggested a low activity of nucleotidase which degrades TMP to thymidine. This limitation was overcome by cloning the TMP phosphohydrolase (TMPase) gene of the unusual bacteriophage, PBS2. As a result, TMP in recombinant cells decreased from 230 mumol g(-1) cell to 20 mumol g(-1) cell with accumulation of 500 mg thymidine l(-1) in the medium.