Bovine serum albumin was found to have an inhibitory effect on acid DNase from rat small intestinal mucosa. The inhibitory activity showed pH-dependency. Thus, the highest inhibition was observed at about pH 4.3 but conversely the enzyme was activated at about pH 4.7. The inhibitory effect was heat-inactivated most strongly at about pH 5, but at more acidic or alkaline pHs, no inactivation was observed. Inhibitory activities of serum albumin of various species were comparable with that of bovine serum albumin. Acid DNases from guinea pig kidney and small intestinal mucosa and from rat spleen and kidney were similarly inhibited by the albumin. The acid DNase displays typical Michaelis-Menten kinetics but the kinetics became sigmoidal in the presence of the inhibitor. With increasing inhibitor concentration, the sigmoidal shape became more pronounced, and at high concentration, the DNA was able to compete with the inhibitor and to reverse its action. Among the cyanogen bromide-cleaved fragments of bovine serum albumin, fragment C (derived from the carboxyl-terminal two-thirds of the albumin) had an inhibitory effect comparable to that of intact bovine albumin, but fragment N (derived from the amino-terminal one-third of the albumin) had no activity. Reduced fragment C showed a markedly decreased effect and lost the activity completely after separation into its three component peptides. Acetylation of bovine serum albumin completely destroyed its inhibitory activity.