The benzylacyclouridines, potent and specific inhibitors of mammalian uridine phosphorylase, were also found to be inhibitors of uridine phosphorylase but not thymidine phosphorylase from Escherichia coli. Competitive inhibition was observed in all cases and the most potent of these compounds was HM-BBAU (5-(3-benzyloxybenzyl)-1-[(2′-hydroxy-1′-hydroxymethyl)methyl]uracil) with a Ki value of 0.15 μM. The inhibitory potencies of these compounds parallel those obtained with enzymes from mammalian sources [Niedzwicki et al., Biochem. Pharmac. 31, 1857 (1982) and Naguib et al., manuscript in preparation] indicating that the structure of the active site of uridine phosphorylase from E. coli may resemble that of the mammalian enzyme. © 1986.