Nucleoside phosphotransferase (nucleotide:3'-deoxynucleoside 5'-phosphotransferase, EC 2.7.1.77) from yellow lupin seedling cotyledons was purified and the active enzyme consists of a single polypeptide chain, Mr = 72 000 +/- 3000. In transphosphorylation, purine and pyrimidine nucleosides are good phosphate acceptors and 5'-nucleotides are effective phosphate donors. Among 2'- and 3'-nucleotides, only 3'-AMP and 3'-psi MP acted as phosphate donors, and p-nitrophenylphosphate appeared less active in this regard. The purine and pyrimidine bases inhibit transphosphorylation. The Km values determined for the inosine:5'-AMP pair were 400 micrometers for both the compounds. The enzyme showed optimum activity at pH 8.0 in mM Tris-HCl buffer. Antisulfhydryl reagents and EDTA did not affect enzyme activity.