According to theory, the action of acetylcholine (ACh) and ACh-esterase is essential for the permeability changes of excitable membranes during activity. It is, therefore, pertinent to know the activity of ACh-esterase per unit axonal surface area instead of per gram nerve, as it has been measured in the past. Such information has now been obtained with the newly developed microgasometric technique using a magnetic diver. (1) The cholinesterase (Ch-esterase) activity per mm(2) surface of sensory axons of the walking leg of lobster is 1.2 x 10(-3) microM/hr. (sigma = +/- 0.3 x 10(-3); SE = 0.17 x 10(-3)); the corresponding value for the motor axons isslightly higher: 1.93 x 10(-3) microM/hr. (sigma = +/- 0.41 x 10(-3); SE = +/- 0.14 x 10(-3)). Referred to gram nerve, the Ch-esterase activity of the sensory axons is much higher than that of the motor axons: 741 microM/hr. (sigma = +/- 73.5; SE = +/- 32.6) versus 111.6 microM/hr. (sigma = +/- 28.3; SE = +/- 10). (2) The enzyme activity in the small fibers of the stellar nerve of squid is 3.2 x 10(-4) microM/mm(2)/hr. (sigma = +/- 0.96 x 10(-4); SE = +/- 0.4 x 10(-4)). (3) The Ch-esterase activity per mm(2) surface of squid giant axon is 9.5 x 10(-5) microM/hr. (sigma = +/- 1.55 x 10(-5); SE = +/- 0.38 x 10(-5)). The value was obtained with small pieces of carefully cleaned axons after removal of the axoplasm and exposure to sonic disintegration. Without the latter treatment the figurewas 3.85 x 10(-5) microM/mm(2)/hr. (sigma = +/- 3.24 x 10(-5); SE = +/- 0.93 x 10(-5)). The experiments indicate the existence of permeability barriers in the cell wall surrounding part of the enzyme, since the substrate cannot reach all the enzyme even when small fragments of the cell wall are used without disintegration. (4) On the basis of the data obtained, some tentative approximations are made of the ratio of ACh released to Na ions entering the squid giant axon per cm(2) per impulse.