1. All 20 tyrosyl residues of enolase from human and pig muscle were nitrated with tetranitromethane in mild conditions; 2-phosphoglycerate and magnesium ions decreased the rate of reaction. 2. Inactivation took place in two steps: (a), nitration of 6 - 10 tyrosine residues of enolase decreased the activity by about 20%, without affecting Km value, molecular weight of pH dependence; (b) on nitration of 12 tyrosyl residues, inactivation was nearly complete, with concomitant profound changes in the enzyme properties. 2-Phosphoglycerate and magnesium ions present together protected significantly against inactivation. 3. The results suggest that the activity of enolase is dependent on intactness of tyrosyl residues.