As determined by atomic absorption, fully activated human liver arginase contained 1.1 +/- 0.1 Mn2+/subunit. Upon dissociation to inactive subunits (< 0.01 Mn2+/subunit), there was decreased intensity and a red shift in the tryptophan fluorescence emission spectra of the enzyme, and the resulting species were markedly sensitive to thermal and proteolytic inactivation by trypsin. Arginine and lysine specifically protected the subunits from heat inactivation. Subunit activation by Mn2+ followed hyperbolic kinetics (Kd = 0.08 +/- 0.01 microM). In addition to Mn2+, Ni2+ and Co2+ converted inactive subunits into active monomers, and favoured their association to the oligomeric state of the enzyme (M(r) = 120,000 +/- 2000). The replacement of Mn2+ by Ni2+ or Co2+ resulted in significant changes in Vmax without any change in the Km values for the substrates (arginine or canavanine) or the Ki value for lysine inhibition. The results support our previous suggestion (Carvajal et al., 1994) that Mn2+ is not essential for substrate binding to arginase, and substantiates the conclusion that species differences may exist in the interaction of arginase with metal ions.