Transcutaneous electrical nerve stimulation (TENS) is a non-pharmacological modality used clinically to relieve pain. Central involvement of serotonin and endogenous opioids are implicated in TENS-induced analgesia. Activation of spinal cholinergic receptors is antinociceptive and these receptors interact with opioid and serotonin receptors. In the current study, the possible involvement of spinal cholinergic receptors in TENS analgesia was investigated in rats. Hyperalgesia was induced by inflaming one knee joint with 3% kaolin-carrageenan and assessed by measuring paw withdrawal latency (PWL) to heat before and 4 h after injection. The non-selective nicotinic antagonist mecamylamine (50 μg), non-selective muscarinic antagonist atropine (30 μg) or one of the muscarinic subtype antagonists: pirenzepine (M 1 , 10 μg), methoctramine (M 2 , 10 μg), 4-DAMP (M 3 , 10 μg), or saline was administered intrathecally just prior to TENS treatment. Low or high frequency TENS was then applied to the inflamed knee and PWL was determined again. Atropine, pirenzepine and 4-DAMP significantly attenuated the antihyperalgesic effects of low and high frequency TENS while mecamylamine and methoctramine had no effects, compared to saline control. The results show that TENS-induced antihyperalgesia is mediated partially by activation of spinal muscarinic receptors but not spinal nicotinic receptors. Further, the results also indicate that spinal M 1 and M 3 muscarinic receptor subtypes mediate the muscarinic component of TENS antihyperalgesia.