5'-Nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) occurs in bull seminal plasma in multiple forms. The heterogeneity does not reflect the existence of true isoenzymes, but is due to the association of the enzyme with particulate material and to molecular aggregation phenomena. Addition of detergents to native bull seminal plasma prevents molecular aggregation, solubilizes the particulate form of the enzyme, and results in the appearance of a single molecular form of the enzyme. Enzyme purification can be achieved after three chromatographic steps which involve negative adsorption of 5'-nucleotidase activity on DEAE-Sephadex A-50 followed by two affinity chromatographies on concanavalin A-Sepharose 4B and ADP-agarose. The enzyme appears to be a dimeric glycoprotein. Some properties of the enzyme, including substrate specificity and the effects of hydrogen ion concentration and of various divalent cations, are reported.