An exocellular pyrophosphatase, active on the nucleotide precursors of peptidoglycans, has been found in the culture medium ofStreptomyces mediterraneiME/R 17. This enzyme was separated from theDD-carboxypeptidase by batchwise adsorption on DEAE cellulose. The pyrophosphatase had no strict substrate requirements, it hydrolyzed various UDP-sugar substrates: UDP-GlcNAc. UDP-MurNAc and UDP-MurNAc peptides, giving rise to the corresponding sugar phosphate and to UMP. The enzyme preparation also contained a 5′-nucleotidase activity and UMP was further split to give uridine. This nucleotidase activity was inhibited by potassium tetraborate. Both cytoplasmic and particulate preparations from cells ofS.mediterraneialso contained a pyrophosphatase activity while only the particulate fractions showed theDD-carboxypeptidase activity.The pyrophosphatase excretion was tested during the growth cycle. The activity of the enzyme showed a constant increase throughout the exponential growth and a stronger increase in the late exponential phase. Such a result could be correlated with a consumption of the nutrients in the culture medium, in fact a relatively poor culture medium had a strong positive effect upon the production of the exocellular pyrophosphatase.