Intracellular microelectrode recordings of acinar cell membrane potentials were made from fragments of the rat lacrimal gland superfused in vitro. The average resting membrane potential was -45 mV. Carbachol and epinephrine produced virtually identical membrane potential changes consisting of an initial hyperpolarization (1 mV), lasting approximately 7 sec, followed by a depolarization of approximately 12 mV. The membrane potential generally returned to prestimulation levels after 2 min of exposure to agonist. The responses to carbachol and epinephrine were blocked by atropine and phentolamine, respectively. Superfusion with media lacking Ca or Cl reduced significantly both the resting membrane potential and the agonist-induced depolarization. The hyperpolarization was increased significantly in the absence of Ca and generally prolonged in the absence of Cl. Superfusion with 10 mM Co had no effect on either the resting membrane potential or the agonist-induced membrane potential changes. The hyperpolarization initiated by agonist was significantly enhanced during superfusion with low K, ouabain or amiloride while the depolarization was significantly reduced during superfusion with low K, amiloride or low Na. Resting membrane potentials during superfusion with low K, amiloride or low Na were not significantly different from control, whereas ouabain caused a small depolarization. It is concluded that muscarinic or alpha adrenergic receptor stimulation initiates a membrane potential change characterized by a hyperpolarization, due to an increased in membrane permeability to K, followed by a depolarization due to an increase in membrane permeability to Na.