Circular muscle strips from the opossum esophageal body obtained 3–5 cm above the esophagogastric junction were suspended in organ baths for measurement of isometric tension. Stimulation of nonadrenergic, noncholinergic (NANC) inhibitory nerves was performed using transmural field stimulation (TMS). During TMS, no mechanical response was elicited. After cessation of the stimulus a short period, also without mechanical response, intervened, and this period is called latency. The latency was followed by the ‘off-contraction. In control preparations, the latency and the amplitude of the ‘off-contraction were 1.47 ± 0.17 s, and 3.8 ± 0.9 mN, respectively. The inhibitor of the L-arginine-nitric oxide (NO) pathway, NG-nitro-L-arginine (L-NNA) concentration-dependently reduced the latency at concentrations > 10-6M (n = 6-7). At the highest concentration of L-NNA (10-4M), ‘off contractions were no longer seen. In 5 out of 7 preparations exposed to L-NNA (10-4M), a small contraction was seen during stimulation, and this contraction was abolished by atropine (10-6M) in all strips. L-NNA concentration-dependently reduced the amplitude of contractions at concentrations > 10-6M (n = 6–7). At 10-4M, the amplitude was reduced to 3 ± 2% of that of the initial contraction. Preincubation with L-arginine (10-5M) had no influence on the latency. The effects of L-NNA on both latency and the amplitude of contraction were antagonized by preincubation with L-arginine (10-5M). Atropine (10-6M) had no effect on the amplitude of the ‘off-contraction in control preparations. In conclusion, the L-arginine-NO-generating pathway plays an important role in determination of latency and therefore in the timing of the ‘off-contraction. The ‘off-contraction seems to be a rebound phenomenon.