The in vitro guinea pig ureter responded to 5 sec trains of electrical stimuli with two contractions; the first an on response (ON) occurred with 0.1 ~ 0.3 sec after the onset o the stimulus train, the second an off response (OFF) occurred 0.2 ~ 1.0 sec after the termination of the stimulus train. Relaxation occurred between the two responses during a time when the stimulus was still being delivered. Longer duration and/or higher frequencies of stimuli within the train were required to elicit the OFF than the ON. Decreasing temperature from 37˚ to 22˚ decreased ON amplitude and increased OFF amplitude. Ca++-free solution, 2 mM EDTA, 1 mM Mn++ or 1μM verapamil rapidly abolished ON. OFF persisted when ON had disappeared by repeated stimulation at 0.12 train per sec. Conversely, caffeine, 50μM and theophylline, 10μM abolished OFF with only slight reduction of ON, and sodium nitroprusside decreased preferentially ON amplitude rather than OFF. Relaxation between ON and OFF was incomplete in low Na+ solution. ON and OFF were not affected by the neural blockers tetrodotoxin, atropine or phentolamine, also pyrilamine and methysergide, and relaxation between ON and OFF was Na+ dependent. Furthermore, ON depends on free Ca++ and OFF is more dependent on bound or stored Ca++.