Using 35 new anticancer agents from the screening program of the National Cancer Institute, a biochemical study of the effects on the respiration and oxidative phosphorylation of rat liver mitochondria and on the respiration of leukocyte, liver, L1210 leukemia and Ehrlich ascites cell suspensions was made. Fifteen of the 35 compounds were potent respiratory inhibitors as defined by 50% inhibition of mitochondrial respiration at concentrations of 110 mol/l or less. The mechanism of respiratory inhibition by the drugs was either a rotenone-, antimycin-, or oligomycin-like effect. One triazine derivative showed some specificity for inhibiting tumor cell respiration in comparison with normal cell respiration. Two naphthoquinone derivatives showed inhibition of respiration in in vivo treatments at chemotherapeutic doses. Data on respiratory effects may assist in the interpretation of the results of in vivo and in vitro screening tests of the drugs, and that in some cases, as with the naphthoquinone derivatives, the effects on respiration could be related to the mechanism of action or the mechanism of toxicity of the drugs.