1. A spectrophotometric procedure is described which specifically measures oxidative phosphorylation in submitochondrial particles in the diphosphopyridine nucleotide-cytochrome b segment of the respiratory chain. It is based on the reduction of exogenous coenzyme Q1 by reduced diphosphopyridine nucleotide catalyzed by phosphorylating submitochondrial particles from beef heart and rat liver. 2. Previously described methods for assaying formation of adenosine triphosphate at the first site of oxidative phosphorylation were found to be unsatisfactory. 3. Both the reduction of coenzyme Q1 and the accompanying formation of adenosine triphosphate in this system are unaffected by antimycin and KCN but are almost completely abolished by rotenone or Amytal. 4. The reduction of coenzyme Q1 by succinate is not accompanied by phosphorylation. 5. A survey of various electron acceptors revealed that oxidative phosphorylation at the first phosphorylation site was only observed if electrons passed through the rotenone-sensitive site. 6. In submitochondrial particles from beef heart, octylguanidine, phenethylbiguanidine, and dicumarol failed to exhibit a clear-cut site specificity of action. 7. Addition of the cold-labile, soluble adenosine triphosphatase (PI) to deficient submitochondrial particles stimulated oxidative phosphorylation at all three sites in the respiratory chain.