Painful stimulation increases spinal cord norepinephrine (NE) in animals, and spinally released NE induces acetylcholine (ACh) release to cause analgesia. The purpose of this study was to determine the relationship between NE and ACh in cerebrospinal fluid (CSF) in sheep and humans during painful stimulation. CSF was sampled in anesthetized sheep before and during electrical nerve stimulation at an intensity sufficient to increase mean arterial pressure 15%-20%. To determine whether spinally released NE caused ACh release by stimulation of alpha(2)-adrenoceptors, seven sheep received intrathecal (IT) idazoxan whereas seven sheep received IT saline before stimulation. To examine the effect of pain on CSF NE and ACh in humans, CSF was sampled in 33 women after at least 4 h of painful labor and in 22 pregnant women without pain. Painful stimulation in sheep increased CSF NE and ACh. IT idazoxan blocked the increase in both NE and ACh. Although mean concentrations of CSF NE and ACh did not differ between parturients with and without pain, there was a significant correlation between NE and ACh concentrations only in those with pain. These data provide evidence in animals for activation of spinal cord noradrenergic-cholinergic systems in response to pain. There is only weak evidence for such activation, however, in women with painful labor.