A new method of isolating human eccrine sweat glands by the repeated dissection of skin biopsies with scissors is described. The success of the technique is attributed to a potential line of weakness between the investing capsule and the surrounding connective tissue, which parts under shear forces. The yield is 20-50 glands per biopsy (5 cm X 0.5 cm). The glands are judged to be viable by: (i) light and electron microscopy; (ii) ATP, ADP and AMP contents of 81.0 +/- 12.7, 13.8 +/- 3.3 and 3.8 +/- 1.0 pmol/gland, respectively (mean +/- S.E.M.), which gave an energy charge of 0.90; (iii) the 28-fold rise in cyclic GMP content and the sevenfold rise in cyclic AMP content effected by treatment for 2 min with 10(-5) M-acetylcholine and for 10 min with 10(-5) M-isoprenaline, respectively; (iv) the rate of [3H]leucine uptake into protein; and (v) the concentration of Neutral Red by the collecting duct. Glands were maintained for 7 days on polycarbonate filters floating on RPMI 1640 tissue-culture medium. After this time the ATP, ADP and AMP contents were 63.2 +/- 7.3, 8.5 +/- 2.2 and 3.5 +/- 0.8 pmol/gland, respectively (mean +/- S.E.M.), which gave an energy charge of 0.90. During maintenance a dilatation of the intercellular spaces developed in both secretory coil and collecting duct. Following maintenance there was a significant rise in the rate of [3H]leucine uptake into protein. Maintained glands demonstrated a fivefold greater accumulation of cyclic AMP in response to isoprenaline than did freshly isolated glands, but there was no comparable maintenance hypersensitivity of cyclic GMP to acetylcholine. This pattern of adrenergic, but not cholinergic, maintenance hypersensitivity matches the known lack of denervation hypersensitivity of human eccrine sweat glands to acetylcholine in vivo.