This report extends earlier research, done with external media at pH 7.2, to new studies at pH 6.4 and 8.4 as well as 7.2, to determine the roles of the protonated and neutral forms of physostigmine (a weak base with pKalpha = 8.2) in causing twitch potentiation of the frog sartorius muscle. Physostigmine, especially at relatively high pH (8.4) and concentration (1.5 mM), considerably blocks excitation. However, the results show in general that physostigmine potentiation increased peak contraction time and thereby indicate that potentiation is occurring in terms of prolongation of the active state. At pH 6.4 and 8.4, 1 mM physostigmine causes no change in the mechanical threshold of K depolarization contractures of toe muscles, as found previously at pH 7.2. Physostigmine increasingly prolongs the action potential as pH rises, i.e., in positive correlation with the twitch potentiation, thus indicating that this electrical change is the prime determinant of the potentiation.