Hexachlorophene (HCP) inhibits both endogenous and exogenous respiration (oxygen uptake) in Bacillus megaterium, without sparing by any of several substrates. The inhibition is maximal when the cells are treated with 8 mug of HCP per mg of cells (dry weight), which corresponds to the minimal lethal dose. Levels as low as 2 mug/mg are inhibitory but not lethal. HCP also inhibits the respiration of isolated B. megaterium membranes and can act on several components of the electron transport chain in the membranes and on soluble enzymes. Although both forms of nicotinamide adenine dinucleotide, reduced form dehydrogenase and malic dehydrogenase are inhibited by HCP, they are less susceptible than is oxygen uptake. The site of maximal sensitivity is nearer the terminal electron acceptor, but the exact location depends on the cytochrome composition of the membranes. If cytochromes b(1), a, and a(3) are present, but not o, HCP inhibits electron transport on the substrate side of cytochrome b(1); if cytochromes b(1), a(3), and o are present, but not a, the inhibition occurs on the oxygen side of cytochrome b(1). Exogenous menadione, an analogue of menaquinone, reverses the inhibition in both circumstances. The primary lethal action of HCP thus appears to be respiratory inhibition at a site within the membrane-bound part of the electron transport chain.