Spleen cell suspensions from AKR and CD-1 mice contain peroxidase activity as determined by guaiacol oxidation. This activity is found predominately in the 20,000 x g pellet fraction of spleen cell homogenates. In the presence of H(2)O(2) and chloride ion at acidic pH, splenic peroxidase mediates the oxidation of d- or l-alanine to CO(2), NH(3), and acetaldehyde. The same reaction mixture without added amino acid can kill both gram-positive and gram-negative bacteria. The conditions for both reactions are similar. Both have an absolute requirement for H(2)O(2) and chloride ion, neither is active at neutral or alkaline pH, and both are inhibited by the sulfonic amino acid taurine. In these aspects, splenic peroxidase is qualitatively similar in its activity to myeloperoxidase (MPO) from polymorphonuclear leukocytes. It is quantitatively different from MPO in that the latter is more potent on a per guaiacol unit basis with respect to both amino acid oxidation and bactericidal activity. Still another quantitative difference is that splenic peroxidase requires 0.1 m NaCl for activity, whereas MPO functions with as little as 0.005 m NaCl. Splenic peroxidase and MPO both appear to differ qualitatively from horseradish peroxidase in that the latter enzyme does not mediate amino acid oxidation.