Bacterial phosphatases of growing organisms can be simply demonstrated by incorporating di–sodium–p–nitrophenylphosphate (pNPP) into suitable media. The phosphatase content of a bacterial suspension can be assessed by incubating a measured amount of it with unbuffered or suitably buffered pNPP solutions. The yellow colour (in alkaline solution) of p-nitrophenol liberated from the substrate by phosphatase can be measured accurately and conveniently in a photoelectric absorptiometer, or estimated in a Lovibond comparator using permanent colour standards. The usefulness of the tests for differentiating between members of certain genera is demonstrated by the reactions of Bact. aerogenes and Bact. cloacae, for which a close relationship between phosphatase production and gelatine liquefaction times has been found. The optimal conditions for hydrolysis of pNPP by the phosphatase of a strain of Bact. aerogenes have been established.