A nucleoside phosphotransferase, which catalyzes the phosphorylation of nucleosides to nucleotides by low energy phosphate esters, has been isolated and purified 500-fold from the membrane fraction of Erwinia herbicola. Its most noteworthy difference from other enzymes of this class is that it is membrane bound and can be isolated and handled only in the presence of a detergent. With a ribonucleoside acceptor, adenosine, the reaction product is exclusively 5'-AMP; with deoxyadenosine, 5'- and 3'-nucleotide products appear in the approximate ratio of 2:1, respectively. The enzyme has no detectable phosphatase activity with the best phosphate donors, 5'-dAMP and 5'-dTMP, and very little with less active donors, such as p-nitrophenyl phosphate. This phosphotransferase should be a useful agent for preparing 5'-nucleotides from unusual synthetic bases.