The effect of cholinesterase inhibition on sweat glands of neonates was explored through the local action of intradermally administered neostigmine. All 15 full-size neonates reacted to concentrations of 10 to 10 while only four of 17 low-birth-weight infants were responsive. This differential responsiveness is highly significant (P = 0.01). Differences are even greater when related to gestational age. In addition, subthreshold combinations of neostigmine and acetylcholine were given to three premature and four mature infants in order to potentiate pharmacologic stimuli. None of the premature infants responded to doses as high as paired half-maximal concentrations of each drug, while all mature infants showed sweating. Potentiation could not be shown even in mature neonates with pilocarpine and acetylcholine. This study shows that sweat glands are responsive to endogenous acetylcholine in mature neonates but not in those born prematurely.