The general anesthetics chloroform and halothane inhibit ATP synthesis in rat liver mitochondria, in the millimolar concentration range (1-12 mM), in parallel with a reduction of respiratory control and the ratio of ATP produced to oxygen consumed. In these effects, halothane and chloroform are similar to classical, protonophoric, uncouplers. The rate of ADP-stimulated respiration or the rate of uncoupler-stimulated respiration is not affected. Like classical uncouplers, halothane and chloroform also stimulate mitochondrial ATPase activity. However, the extent of stimulation by these agents is larger than by protonophoric uncouplers and, more significantly, ATPase activity stimulated by carbonylcyanide m-chlorophenylhydrazone is further stimulated by these agents. In the presence of the Ca2+ chelator EGTA, halothane and chloroform have no measurable effect on the magnitude of the proton electrochemical potential, delta mu H. In the absence of EGTA these anesthetics have a small effect on delta mu H, apparently due to stimulation of Ca2+ cycling. Under these conditions the membrane potential is decreased while delta pH is increased, but the total value of delta mu H is only slightly decreased. The uncoupling activity of the anesthetics is the same in the presence of absence of EGTA. Thus, in contrast to protonophoric uncouplers, the uncoupling effect of general anesthetics does not depend on the collapse of delta mu H. In the same concentration range in which anesthetics uncouple oxidative phosphorylation both halothane and chloroform increase membrane fluidity, as measured by the partitioning of the hydrophobic spin probe 5-doxyldecane. These findings suggest a role for intramembrane processes in energy conversion that is not dependent on the bulk delta mu H.