Total activity of the cholinesterases (ChE) and the concentration and output of acetylcholine (ACh) were measured in isolated chicken hearts. The ACh concentration of the right ventricle was as high as that of the total heart (approximately 7 nmol/g). The ChE activity and its kinetic parameters did not differ from those of other vertebrates. In the absence of physostigmine, stimulation of both vagus nerves (20 Hz, 5 minutes) caused an output of ACh and a decrease in rate (-90% of control) and amplitude (-74%) of the ventricular contraction. Physostigmine (10(-7) M) inhibited the ChE by 87%, caused a small reduction of the ACh concentration (P = .05) and more than doubled the ACh output in response to vagal stimulation. In contrast, 10(-6) M physostigmine increased significantly the ACh concentration by about 40% within 5 minutes; the output was almost 3-fold higher than that in the absence of physostigmine and the ChE was nearly completely inhibited (-96%); the output of ACh in the 5th minute of stimulation was not significantly lower than that in the 1st minute. Physostigmine, in a concentration of 10(-4) M, did not change the ACh content of the heart and was much less effective in elevating the ACh output than was a concentration of physostigmine of 10(-6) M. In conclusion, the high ACh output from isolated chicken hearts evoked by preganglionic nerve stimulation was at least partially due to the pronounced innervation of the avian ventricles. Inhibition of the ChE by 96% failed to cause ganglionic transmission block. The formation of surplus ACh required higher physostigmine concentrations (10(-6) M) than the inhibition of the external ChE (protection of ACh released). High physostigmine concentrations (greater than 10(-6) M) counteracted the formation of surplus ACh.