In isolated, intact liver mitochondria from hyperthyroid rats, the L-glycerol-3-phosphate binding site(s) of the L-glycerol-3-phosphate dehydrogenase was (were) found to be influenced by the nature of the electron acceptor, as well as by the pH and the presence of calcium ions. With the hydrophobic electron acceptor menadione a single L-glycerol-3-phosphate binding site was detected kinetically at bulk pH values between 6.5 and 9.0. With the hydrophilic phenazine methosulfate, on the other hand, two L-glycerol-3-phosphate binding sites were distinguishable at pH > or = 7.5 and pH > or = 7.0, in the presence and absence of Ca2+, respectively. The kinetic mechanism of the reaction catalyzed by L-glycerol-3-phosphate dehydrogenase is ping pong Bi Bi with a hydrophilic electron acceptor, where as with the hydrophobic substance, a sequential Bi Bi mechanism was observed. We suggest that the latter mechanism has physiological relevance.