The nucleoside phosphotransferase of barley was isolated in four steps from dried malt sprouts and was obtained homogeneous according to several criteria. The enzyme transfers the phospho group from the 3'- or 5'-position of a nucleotide to the 5'-OH group of another nucleoside. Activated phosphomonoesters (e.g. 4-nitrophenyl phosphate) can be used as donors as well. In the absence of nucleosides the enzyme shows pronounced phosphatase activity towards 3'- and 5'-nucleotides as well as towards activated phosphomonoesters. 2'-Nucleotides are not hydrolysed. Studies of the phosphatase activity with a number of modified nucleotides demonstrate that the nucleotide substrates are bound by the phosphate group and by the base. The base can be extensively modified compared to naturally occurring nucleobases. Obviously, only a hydrophobic interaction with the enzyme is required; base-specific hydrogen bonds are of no importance. In the transferase reaction the acceptor nucleoside interacts only with the base which has a much more restricted specificity. Nucleotides which are donors do not need to be acceptors when they are nucleosides, while all acceptor nucleosides are efficient donors. Due to the pre-orientation of the base, the phosphate groups of the 3'- and 5'-nucleotides are in different positions relative to the groups of the enzyme that are involved in the catalysis. This is reflected in the kinetic parameters which are interpreted on the basis of a mechanism with a phospho-intermediate as postulated for the enzyme of Ives et al. [(1979) J. Biol. Chem. 254, 4339; (1982) J. Biol. Chem. 257, 4931]. Evidence is obtained that Km represents the ratio of the rate constants for the decay and the formation of the intermediate and that the kcat/Km-value can be used as indicator for the formation step.