Efrapeptins, peptides isolated from the fungus Tolypocladium niveum, showed toxic activity, as a crude mixture of five closely related compounds, against twospotted spider mite, Colorado potato beetle,southern armyworm, and tobacco budworm. The insecticidal activity of pure efrapeptins was characterized using two of the compounds against Colorado potado beetle in a foliar spray assay. Efrapeptin F (LC50 = 8.4 ppm) was about twice as potent as efrapeptin D (LC50 = 18.9 ppm). ATPase inhibitory activity of the efrapeptins was demonstrated using mitochondrial preparations from two fungal species, Metarhizium anisopliae and T. niveum, and from an insect, house fly. Against house fly mitochondria, efrapeptin D was the most potent ATPase inhibitor of five efrapeptins tested.