The treatment of abscess homogenates with calcium ionophores stimulated the production of a bactericidal lipid with properties indistinguishable from those of a previously unidentified bactericidal lipid that had been detected in staphylococcal abscesses. The lipid was identified as a monoglyceride by thin layer chromatography. It resembled the unidentified lipid in that it had a high specific activity, exhibited differential activity, was inhibited by Staphylococcus aureus delta toxin, lecithin and Ca++, and its activity was reduced by oxidation. Stimulation of monoglyceride production by calcium ionophore requires the joint presence of components from the sedimented and supernatant fractions of abscess homogenates, and was not produced if boiled homogenate was used. The addition of verapamil interfered with the production of monoglyceride in homogenates treated with calcium ionophore. Monoglyceride was produced only in abscess homogenates and not in homogenates of other normal tissues or tissues taken from mice infected with S. aureus. Calcium ionophore could be replaced by inositol triphosphate, suggesting that monoglyceride production involved the release of calcium from intracellular stores. The 2-monoglyceride was the form originally produced in abscess homogenates, but this spontaneously isomerized to the 1-monoglyceride. The fatty-acid moiety of the monoglyceride consisted primarily of 16:0 and 16:1 fatty acids.