1. The accumulation of [3H]methyltriphenylphosphonium by isolated fat-cells was used to estimate the membrane potential of mitochondria in situ. 2. An alpha-adrenergic receptor-mediated decrease in the apparent accumulation of [3H]methyltriphenylphosphonium was observed. Methoxamine, clonidine and low concentrations of phenylephrine decreased the calculated mitochrondrial membrane potential without significantly raising cyclic AMP levels, adenylate cyclase activity or stimulating lipolysis. The agonist potency order was phenylephrine greater than methoxamine greater than clonidine. 3. The decrease in the calculated mitochondrial membrane potential caused by phenylephrine, clonidine and methoxamine was blocked by the alpha-adrenergic antagonist prazosin but not by yohimbine nor by the beta-antagonist propranolol. This suggests that the effect on the calculated mitochondrial membrane potential may be mediated by alpha 1-like receptors.