We examined human tears for molecules that killed gram-positive bacteria. The principal mediator of bactericidal activity against staphylococci proved to be a calcium-dependent enzyme, secretory phospholipase A₂. Whereas the concentration of secretory phospholipase A₂ in the normal tear film exceeded 30 μg/ml, only 1.1 ng (<0.1 nM) of the enzyme per ml sufficed to killListeria monocytogenes and 15 to 80 ng/ml killedStaphylococcus aureus. Despite its efficacy against gram-positive bacteria, secretory phospholipase A₂ lacked bactericidal activity against gram-negative organisms (Escherichia coli, Salmonella typhimurium, andPseudomonas aeruginosa) when tested in the ionic environment of tears. Given the presence of secretory phospholipase A₂ in tears, intestinal secretions, and leukocytes, this enzyme may play a substantial role in innate mucosal and systemic bactericidal defenses against gram-positive bacteria.