Constitutive arginase (molecular weight 330,000) 920-fold purified from Evernia prunastri thallus, is activated by putrescine, l-ornithine, and agmatine with K(a) values of 2.7, 1.1, and 5.8 millimolar, respectively. Constitutive arginase is also activated by endogenous l-arginine, reaching its maximum activity at 16 hours of incubation on Tris-HCl (pH 9.15) with a subsequent decrease. Urea behaves as a mixed inhibitor of the enzyme with a K(i) value of 2.6 millimolar. Atranorin and evernic acid behave as in vitro activators of the enzyme; usnic acid does not have any significant effect as activator.