Electrochemical reduction of p-benzoquinone (BQ) across the benzonitrile (BN) layer was investigated in 0.1 M phosphate buffer. Using the method reported by Shi and Anson (Anal. Chem. 70 (1998) 3114), we produced a modified electrode with a thin BN layer of 200 m thickness containing 0.1 M tetrabutylammonium perchlorate. We could observe a two-step reduction process of BQ in the absence of lipid molecules in the BN layer, like that recorded in aprotic solvents. However, introducing 1.0 mM dilauroyl phosphatidylcholine (PC) in the BN layer, the redox response of BQ accompanying the formation of the hydrated form of BQ could be observed as it was formed in aqueous solution simultaneously with the two-step reduction of BQ. Judging from the pH dependence of the redox potential, we believe that HBQ(.) was generated in the reduction of BQ with dilauroyl-PC. This indicates that the hydrated form of the phosphate group (-P(O)OH) provides H+ on the reduction of BQ even in BN. Thus, the function of dilauroyl-PC to carry H+ in the lipophilic atmosphere has been clarified. The same function was found in the case of dipalmitoyl-PC, while the redox response involving the hydrated form of BQ was not observed with diarachidoyl-PC, which has longer alkyl chains.