Pseudomonas aeruginosa phosphorylcholine phosphatase from periplasmic extracts of bacteria grown on choline as the sole carbon and nitrogen source was purified to homogeneity. The enzyme represented nearly 1% of the total protein found in the periplasmic space and is a monomer of approximately 53 kDa with an isoelectric point of 7.5. The optimum pH with phosphorylcholine was in the range of 5-8; with phosphorylethanolamine there was a peak at pH 6, and with p-nitrophenyl-phosphate (p-NPP) the optimum was at pH 5. Studies carried out at pH 5 indicated: i) For the three substrates above, Mg2+, Zn2+, or Cu2+ was necessary for maximal activity. ii) With p-NPP, these cations bound to the free enzyme in an ordered bireactant system. iii) With phosphorylethanolamine, Mg2+, Zn2+, or Cu2+ bound to the free enzyme in an at random bireactant system, iv) With phosphorylcholine, maximal activity was obtained with cation concentrations as low as 100 nM. v) Al3+ ions were inhibitors of the enzyme activity. The n (Hill coefficient) values for the inhibition by Al3+ with phosphorylcholine or p-NPP were 1 or 4, respectively. vi) The enzyme exhibited two affinity sites for phosphorylcholine. With Mg2+, a site with a K-m value of 0.5 mM was detected; the corresponding V-max was 25 mu mol min(-1) (mg protein)(-1); without Mg2+, the enzyme displayed K-m and V-max values of 0.09 mM and 4.2 mu mol min(-1) (mg protein)(-1), respectively. Studies carried out at pH 7.4 indicated: i) The enzyme could not catalyze the hydrolysis of p-NPP, and phosphorylethanolamine was a poor substrate in either the presence or absence of divalent cations. ii) The enzyme activity measured with phosphorylcholine was independent of divalent cations or was not inhibited by Al3+ ions. iii) With or without Mg2+, the enzyme exhibited two affinity sites for phosphorylcholine; the K-m values were 0.05 mM and 0.5 mM with their corresponding V-max of 5.6 and 25 mu mol min(-1) (mg protein)(-1), respectively.