A procedure is proposed for the separation of multiple forms of 5'-nucleotidase (EC 3.1.3.5) by cellulose acetate electrophoresis. The effects of various treatments (wheat-germ lectin, neuraminidase, n-butanol, papain, Triton X-100 and precipitation of LDL and VLDL) on the electrophoretic pattern of 5'-nucleotidase and alkaline phosphatase were studied. In healthy controls, the presence of three fractions with alpha 1, alpha 2 and beta mobilities was observed, the latter being the major one. In different hepatobiliary diseases a close relationship between the presence of high molecular weight alkaline phosphatase and the increase in the ratio Total/beta 5'-nucleotidase was observed, showing that the increase in total 5'-nucleotidase in these patients is mainly due to the alpha 1 isoform. The nature of these forms is discussed.