Isometric tension developed by denervated soleus and extensor digitorum longus (EDL) muscles in vitro in response to direct electrical stimulation and to acetylcholine (ACh) was measured. The effect of β-endorphin and immunologically related peptides on these responses was investigated and compared to those of opiate receptor agonists. β-Endorphin reversibly augmented contracture responses to ACh by up to 2.4-fold. β-Lipotropin was as potent as β-endorphin in augmenting the ACh response by 2-fold, whilst lipotropin-(61–87)(C′-fragment) was over 100 times less potent. The α-N-acetyl derivatives of β-endorphin and the C′-fragment were not effective in altering ACh responses, and neither were β-MSH and ACTH-(1–39). α-Endorphin, [Leu 65]β-endorphin, enkephalins and opiate narcotic agonists were effective, but were considerably less potent than β-endorphin, causing a maximum augmentation of the ACh contractures of only 1.4–1.7-fold. Tissue culture media which had been incubated with immature rat embryonic spinal cord explants mimicked the effect of β-endorphin on ACh contracture responses. A high concentration of the opiate antagonist naloxone (10 −4 M) reduced the ACh contractures. At lower (10 −6–10 −5 M) concentrations, naloxone alone was not effective but it partially prevented the augmentation of contratuures produced by both β-endorphin and spinal cord conditioned culture media. Neither the peptide nor conditioned media had any effect on responses of the muscles to electrical stimulation and neither augmented responses to carbachol, a cholinoceptor agonist which is not hydrolysed by acetylcholinesterase. β-Endorphin did not increase the ACh response in muscles which had been pretreated with neostigmine, but it caused a slight increase in the responses in muscles pretreated with edrophonium chloride which is a weaker anticholinesterase agent. Major molecular forms of acetylcholinesterase in crude muscle extracts were separated by centrifugation on sucrose density gradients and their activity was measured in EDL and soleus muscles which had been denervated six days previously and in muscles from unoperated limbs. Although their activity was diminished in denervated muscles, all major forms including the 16S (‘endplate-specific’) form were present after denervation as in muscles with intact innervation. It was concluded that the action of β-endorphin and related peptides in augmenting the ACh contracture responses was probably due largely to their anticholinesterase activity.