5'-Nucleotide phosphodiesterase (5'-NPDase) was partially purified from plasma membranes of murine lymphoma L5178Y. The enzyme was inactivated by N-ethylmaleimide and Zn2+, but stabilized by dithiothreitol, suggesting that it is an SH enzyme. The enzyme, Km 1.54 mM, pI 5.8 and MW 23k, differs from liver 5'-NPDase in MW, Km and sensitivity to some inhibitors. On the contrary, 5'-NPDase, derived from normal mouse organs, is similar to the liver enzyme. The results suggest that tumor cells possess a novel molecular species of 5'-NPDase.