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In Bacillus subtilis 168 a class of point mutations (locus “phoP”) brings about the simultaneous loss of alkaline phosphatase and of a “phosphodiesterase” [substrate bis(p-nitrophenyl)phosphate] activities. The latter enzyme also hydrolyzes UDPG and (the phosphatase substrate) p-nitrophenyl phosphate. In order to test a possible structural relationship between them, the two enzymes were purified and their subunit composition was studied. A new purification procedure for the alkaline phosphatase was developed; it leads to the isolation of pure enzyme with high yield. A modification of this procedure gave highly purified (but not homogeneous) preparations of “diesterase”. The molecular weights of the two enzymes are different: alkaline phosphatase 75000, “phosphodiesterase” 150000. Each enzyme is composed of two subunits of identical size; the subunits of the “diesterase” being about double in size of those of the phosphatase. The enzymes also differ in their pH optimum for p-nitrophenyl phosphate (alkaline phosphatase pH optimum 10.2, “phosphodiesterase” pH optimum 8.0). The amino acid composition of the alkaline phosphatase was determined; the enzyme molecule is rich in basic amino acids (especially lysine). Apparent Km (for p-nitrophenyl phosphate) (= 0.2 mM at pH 10.00) and Ki (for Pi) (= 0.37 mM at pH 10.0) were measured for the purified alkaline phosphatase. Incorporation of [14C]isoleucine into proteins during Pi starvation followed by isolation of the labelled alkaline phosphatase showed that the enzyme is synthesized de novo in these conditions. The results presented indicate that (a) the enzymes are indeed repressed in presence of excess Pi and (b) they are not structurally related and, especially do not share a common subunit. The “phoP” mutations do not therefore affect a structural gene for these enzymes. Hypotheses concerning the function blocked in these mutants are discussed. [ABSTRACT FROM AUTHOR]