The endotoxin lipopolysaccharide (LPS) from Gram-negative bacteria exerts a direct and rapid effect on tissues. While most attention is given to the downstream actions of the immune system in response to LPS, this study focuses on the direct actions of LPS on skeletal muscle in Drosophila melanogaster. It was noted in earlier studies that the membrane potential rapidly hyperpolarizes in a dose-dependent manner with exposure to LPS from Pseudomonas aeruginosa and Serratia marcescens. The response is transitory while exposed to LPS, and the effect does not appear to be due to calcium-activated potassium channels, activated nitric oxide synthase (NOS), or the opening of Cl− channels. The purpose of this study was to further investigate the mechanism of the hyperpolarization of the larval Drosophila muscle due to exposure of LPS using several different experimental paradigms. It appears this response is unlikely related to activation of the Na-K pump or Ca2+ influx. The unknown activation of a K+ efflux could be responsible. This will be an important factor to consider in treatments of bacterial septicemia and cellular energy demands.