Mechanical function and mitochondrial respiration were observed in newborn pig hearts in the presence of chloramphenicol. Isolated perfused hearts exposed to chloramphenicol (25, 50, or 100 micrograms/ml) demonstrated acute reductions in pressure development and cardiac output accompanied by elevated left atrial filling pressure. The effects of chloramphenicol and chloramphenicol succinate (25, 50, 100, or 200 micrograms/ml) on oxidative activity of isolated mitochondria were also investigated. With unesterified chloramphenicol, inhibition of state 3 respiration was most apparent when glutamate and palmitylcarnitine were supplied as substrates. Inhibition of mitochondrial oxidation of succinate or glutamate was only detectable at 200 micrograms/ml. Inhibition of alpha-ketoglutarate oxidation was not seen at any concentration of antibiotic studied. Chloramphenicol succinate most strongly inhibited state 3 oxidation of succinate and alpha-ketoglutarate and had relatively mild effects on oxidation of glutamate and palmitylcarnitine. Succinate oxidation by submitochondrial particles was unaffected by chloramphenicol succinate, a result suggesting interference with succinate transport.