Physostigmine-induced contractions of isolated small bundles (100 micron diameter) of muscle fibres isolated from the diaphragm of C57 BL mice were studied under various conditions. At different external pH the changes in the maximal amplitude of the contraction were related to the external activity of the permeant neutral form of the drug. However changes in the internal pH showed that the amplitude of the contractile response depended directly on the internal activity of the protonated form of the drug. The amplitude of the contraction was enhanced by caffeine treatment and depended on the external calcium concentrations. In mammalian skeletal muscle, physostigmine appeared to produce contractions due to its anticholinesterase property. The release of calcium seemed to occur from an intracellular store different from that involved in the action of caffeine.