Three periplasmic nucleoside monophosphate-splitting phosphatases from Salmonella typhimurium, 2':3'-cyclic nucleotide 2'-phosphodiesterase, nonspecific acid phosphatase I, and nonspecific acid phosphatase II, were separated by column chromatography. They are characterized with respect to their substrate specificities, Km values, pH optima, molecular weights, and sensitivity to inhibition by Pi and EDTA. Nonspecific acid phosphatase II has not been reported previously. The physiological roles of the various phosphatases are assessed by studies on mutant strains. Selection procedures were developed for the isolation of mutants defective in the synthesis of one or more of the phosphatase activities. Analysis of these mutant strains revealed that 2':3'-cyclic nucleotide 2'-phosphodiesterase is the major 3'-nucleotide dephosphorylating activity, and nonspecific acid phosphatase II is most active against 5'-nucleotides at concentrations below 1 mM. Triple mutants, lacking all periplasmic nucleotide-splitting activities, are viable. Regulatory mutants with elevated levels of nonspecific acid phosphatase II activity were isolated. The gene for 2':3'-cyclic nucleotide 2'-phosphodiesterase (pde) was located between purA and argI at 135 min on the S. typhimurium linkage map.