Transmitter release from frog motor nerve terminals occurs at specialized sites on the nerve terminal called active zones (AZs). We have used a low calcium (0.1 nM) saline treatment to disrupt AZ structure and correlated these changes with alterations in transmitter release from the nerve terminal. Exposure to 0.1 nM free calcium saline for 3 h caused many individual AZs to break into two or three pieces, apparently unorganized particles drifted free of the AZ array, and the normally ordered alignment of AZ particles was loosened. Despite these forms of disruption in AZ organization, physiological function remained remarkably normal. Although the size of the endplate potential recorded in response to a single nerve stimulus was little affected, paired-pulse facilitation and tetanic potentiation were significantly increased. Synaptic depression was not apparent during the tetanus, but was revealed following the cessation of the stimulation. The results are consistent with the hypothesis that 0.1 nM calcium treatment detached AZ segments from the anchoring molecules that normally hold these proteins in alignment with other synapse-specific molecules. We propose that the ordered AZ organization serves to bring the calcium channels that regulate transmitter release in close proximity to other proteins that are critical to the modulation of release, especially during periods of high frequency stimulation. We hypothesize that the drifting AZ segments, although capable of apparently normal transmitter release, may not be tightly coupled with the intracellular calcium handling proteins that normally restrict the time that calcium ions have to act on the transmitter release apparatus following each action potential.