Seven synthetic polymers, (Glu4, Tyr)n, (Arg)n, (Arg, Pro, Thr)n, (Arg-Gly-Glu)6, (Arg-Gly-Phe)6, (Glu-Arg-Gly-Phe)5, and (Ala-Leu-Arg-Arg-Ile-Arg-Gly-Glu-Arg)2, were treated with phosphoryl chloride to phosphorylate their Tyr, Thr, and Arg residues. Protamines and histones were phosphorylated similarly. These phosphorylated peptides were examined as to whether or not they serve as substrates for intestinal alkaline phosphatase [EC 3.1.3.1] and liver N(omega)-phosphoarginine phosphatase [Kuba, M., Ohmori, H., and Kumon, A. (1992) Eur. J. Biochem. 208, 747-752]. Phosphorylated polyarginine was hydrolyzed with a lower Km with alkaline phosphatase than with N(omega)-phosphoarginine phosphatase, while the phosphorylated forms of (Arg-Gly-Phe)6 and culpeine were better substrates for N(omega)-phosphoarginine phosphatase. When (Arg, Pro, Thr)n and culpeine were phosphorylated chemically after treatment with phenylglyoxal, these phosphorylated peptides were worse substrates for N(omega)-phosphoarginine phosphatase than for alkaline phosphatase. Moreover, the results of proton-decoupled 31P NMR analysis indicated that N(omega)-phosphoarginine phosphatase released Pi from N(omega)-phosphoarginine residues of phosphopeptides. These results indicate that both phosphatases function as protein arginine phosphatases in different manners, and that N(omega)-phosphoarginine phosphatase is useful for selectively detecting N(omega)-phosphoarginine residue in peptides containing various kinds of phosphorylated amino acids.