Bovine pancreatic deoxyribonuclease requires divalent metal cations for hydrolysis of DNA. The effects of calcium and magnesium, alone and combined, on the rate and kinetics of the reaction were examined. Divalent metal salts of DNA were used as substrates. The ratio of either Ca-2+ or Mg-2+ to DNA-P in these salts was 1:2. The Mg-2+ salt of DNA was found to have sufficient Mg-2+ for optimal DNAase activity. Addition of MgCl-2 to a large excess of Mg-2+ over DNA-P had no effect on the rate. Km for the hydrolysis of Mg-2+-DNA was 1.76 mM. Km for the hydrolysis of Ca-2+-DNA was too low to measure by our methods of assay, indicating a high affinity of enzyme for substrate. The rate of hydrolysis of Ca-2+-DNA, however, is slow compared to that of Mg-2+-DNA. By mixing the Ca-2+ and Mg-2+ salts of DNA, a synergistic effect on the activity of DNAase was observed. On the basis of kinetic studies the synergistic effect is attributed to an increased affinity of DNAase for DNA (Km equals 0.34 mM for the mixed Ca-2+, Mg-2+ salt of DNA). A 2-fold increase in DNAase activity was observed when DNAase was incubated in CaCl-2 before assay. This 2-fold stimulation was not related to the synergistic effect. DNAase I is inactive against the sodium salt of DNA. In experiments with mixed Na+ and Mg-2+ salts of DNA, Na+-DNA was found to be a competitive inhibitor of the action of DNAase against Mg-2+-DNA.