This proposal is written in response to an RFA regarding sex and gender- related differences in pain and analgesic response. Others have noted minor differences between the sexes in analgesic responses to systemically administered drugs, with most work being performed with opioids. We note in preliminary experiments a 2-fold increase in potency of intrathecal neostigmine in women volunteers and patients compared to men. Frequency and intensity of side effects are not different between men and women. Positron emission tomography (PET) studies in rhesus monkeys support an estrogen-dependent increase in spinal cholinergic activity in females, and studies in rats demonstrated a 10-fold increase in antinociceptive potency of intrathecal neostigmine and a nicotinic receptor subtype selective agonist, and in potency of systemically administered physostigmine in females. This sex difference in analgesic potency applies equally to acute nociceptive responses as well as subacute and chronic models of hyperalgesia and allodynia. We propose a 3 year series of in vitro and in vivo studies in rats, monkeys, and humans to establish the magnitude and pharmacology of this increased sensitivity to cholinociceptive agents in females and to examine pharmacologic and neurobiologic mechanisms responsible for this increased sensitivity. Specifically, we will test whether this sex difference reflects altered neurotransmitter release, postsynaptic receptor number, second messenger generation, or neurotransmitter interactions. Since intrathecally administered neostigmine has already demonstrated efficacy in clinical trials, and since nicotinic receptor subtype selective agents and novel cholinesterase inhibitors for systemic administration are under active clinical development for other indications, these studies will be instrumental to the understanding and development of systemic and intrathecal therapy for acute and chronic pain in women.