The susceptibilities of three Gram-positive cocci to medium-chain saturated and long-chain unsaturated fatty acids and their one-monoglycerides were studied. The bacteria were incubated with equal volumes of lipid solutions for 10 min. Lauric acid, palmitoleic acid and monocaprin reduced the number of CFU by 6.0 log10 or greater at 5 mM concentration for streptococci of group A (GAS) and group B (GBS). When further compared at lower concentrations and after longer incubation time monocaprin proved to be the most active. Capric acid showed the highest activity against Staphylococcus aureus at 10 mM. However, at lower concentrations monocaprin was the only lipid that showed significant activity against S. aureus. The mode of action of monocaprin against GBS was studied by a novel two-color fluorescent assay of bacterial viability and by electron microscopy. The results indicate that the bacteria are killed by disintegration of the cell membrane by the lipid, leaving the bacterial cell wall intact. The highly lethal effect of monocaprin indicates that this lipid might be useful as a microbicidal agent for prevention and treatment of infections caused by these bacteria.