Abstract Nucleoside phosphotransferases were found in a large variety of plant and animal tissues. In plants and bacteria, only 5′-nucleotides are formed, while in animals small amounts of the 3′-isomers are also produced. The rates of phosphate transfer from phenylphosphate and 5′-inosinic acid are of the same order of magnitude in plants, bacteria and protozoa, but in the mammals transfer from the nucllotide is much slower. The enzyme is unevenly distributed in the organs of the rat, with the highest activities in liver, spleen and kidney, and the lowest in muscle. The bulk of the enzymic activity is localized in the cytoplasmic particles. Considerable increases in the activity of the enzyme occur during germination of wheat, regeneration of rabbit muscle, and bacterial growth. The results point to an active role of the nucleoside phosphotransferases in nucleotide biosynthesis.