This paper describes a method for isolating and studying the metabolism of human eccrine sweat glands. (a) Electron microscopy of glands which had been isolated and then incubated for an hour revealed no apparent alteration in morphology. (b) Known variation in gland size (male > female > children) was reflected in the relative rates of lactate production. (c) Lactate production was approximately 1.5 nmoles/gland per hr in the absence of glucose and rose to 2.7 at physiological concentrations of glucose (5.6 mmoles/liter). This amount of lactate production agrees well with the amounts found in sweat. (d) Both adrenergic (epinephrine) and cholinergic (methacholine) stimuli increased lactate production. (e) Glycogen depletion was demonstrated during incubation. (f) O(2) consumption was measured and aerobic metabolism was found to account for less than 1% of the energy derived from anaerobic pathways. These studies demonstrate that the large amounts of lactate appearing in human eccrine sweat can be accounted for by glandular metabolism and that both glycogen and glucose can be used as substrates.