The in vitro guinea pig ureter responds to 5-s trains of electrical stimuli with two contractions: the first, an "on" response, occurs within 0.1-0.3 s after the onset of the stimulus train; the second, an "off" response, occurs 0.2-1.0 s after the termination of the stimulus train. Force decreases between the two responses during a time when the stimulus is still being delivered. Longer duration and/or higher frequencies of stimuli within the train are required to elicit the off response than the on response. Neither the on nor the off response appears to be neurally mediated, since both responses are unchanged by tetrodotoxin, phentolamine, atropine, and pyrilamine. Decreasing temperature from 37 to 22 degrees C decreases the amplitude of the on response and increases the amplitude of the off response. Calcium-free solution, 2 mM ethylenediaminetetraacetic acid (EDTA), 1 mM Mn2+, and 1 microM verapamil abolish the on response at a time at which the off response continues to persist. Conversely, 0.5 mM caffeine and 0.1 mM theophylline abolish the off response, whereas they only slightly reduce the on response. These data suggest that the on response depends on extracellular free calcium, whereas the off response is more dependent on bound or stored calcium.