The 5'-nucleotidase activity of the purified cytoplasmic fraction preparation of bovine brain does not depend on the presence of the divalent metal ions Mg2+, Ca2+, and Cu2+ in the incubation medium. The Zn2+ ion (0.5 mM) causes total enzyme inhibition. Although EDTA and 8-hydroxyquinoline inhibit the 5'-nucleotidase from this source, it has not been possible to show the existence of metal ions in the enzyme molecule. The inhibition of 5'nucleotidase by EDTA is progressive and irreversible; when the enzyme is not preincubated with EDTA, the inhibition is overridden by metal ions. The purines (except xanthine, 0.3 mM), pyrimidines, and their nucleosides do not affect the 5'-nucleotidase activity. The nucleoside di- and triphosphates are competitive enzyme inhibitors against 5'-AMP as substrate. The Ki values of the diphosphates are lower than those determined for the corresponding triphosphates. The inhibition caused by the above nucleotides is reversed, partly or wholly, by Mg2+, depending on the molar ratio between the effectors. The inhibitory action of the -SH group reagents on the 5'-nucleotidase activity is weak and reversible.