β-Adrenergic-stimulated sweat secretion is mediated by calcium and cAMP. We describe here, for the first time, protein kinase A- (PKA) and protein kinase C- (PKC) dependent modulation of intracellular calcium ([Ca²⁺]_i) by the hormone 17 β-oestradiol (E₂) in the human eccrine sweat gland cell line NCL-SG3. We have previously shown that E₂ rapidly activates PKC isoforms and PKA in NCL-SG3 cells (1). Changes in [Ca²⁺]_i were measured using ratiometric fluorescence microscopy on cells loaded with Fura 2-AM. Variations in [Ca²⁺]_i are expressed as ratios of fluorescent intensities F(t)/F(t0) where F(t) refers to the Fura 2 fluorescence intensity at a given time (t) and F(t0) to fluorescence intensity at zero time basal levels. Cells treated with thapsigargin responded with a transient single spike-like increase in calcium, which then returned to zero time basal levels. Exposure of cells to both E₂ and thapsigargin produced a secondary, sustained increase in [Ca²⁺]_i (F(t20)/F(t0) = 2.68 ± 0.15; n=8 compared with thapsigargin treatment alone, F(t20)/F(t0) = 1; n=8). This effect was independent of extracellular calcium and is specific to 17β oestradiol, as 17α oestradiol and testosterone had no effect on [Ca²⁺]_i. The E₂-induced increase in [Ca²⁺]_i was abolished by the PKC inhibitor chelerythrine chloride (1μM), by the PKA inhibitor Rp-adenosine 3′,5′-cyclic monophosphorothioate (200μM), and by the adenylyl cyclase inhibitor SQ22536 (200μM). Xestospongin C (10μM), a membrane-permeable selective inhibitor of IP₃-induced Ca²⁺ release and IP₃ receptor-mediated signalling, failed to abolish the E₂-induced increase in [Ca²⁺]_i. However, ryanodine (100μM), which at high concentrations is an established inhibitor of ryanodine receptors, completely abolished the E₂-induced increase in [Ca²⁺]_i. These results demonstrate a novel mechanism by which E₂ modulates [Ca²⁺]_i via a PKC-PKA-dependent pathway from intracellular Ca²⁺ stores regulated through ryanodine receptors.