The release of intracellularly stored calcium (Ca2+) contributes to the rise in cytoplasmic Ca2+ concentration during agonist-induced activation of airway smooth muscle. We describe a novel preparation--human airway smooth muscle cells cultured in monolayers--which has enabled the investigation of the identity and the function of this intracellular Ca2+ store. Cells, enzymatically dispersed from surgically resected human bronchi, were cultured in monolayers and confirmed as smooth muscle by positive immunocytochemical staining for actin and myosin. The release of intracellularly stored Ca2+ in response to bronchoconstrictor agonists, histamine and carbachol, was demonstrated by stimulation of Ca2+ efflux from 45Ca-labelled cells. The technique of permeabilisation of the plasmalemmal membrane by saponin allowed the measurement of the Ca2+ content of the intracellular store in 45Ca-labelled cells. Uptake of Ca2+ by the store was energy-dependent and was enhanced by cyclic AMP. The effects of inhibitors of sarcoplasmic reticulum and mitochondrial function on Ca2+ uptake identified the store as sarcoplasmic reticulum. Inositol 1,4,5-trisphosphate released stored Ca2+ in a time and dose-dependent manner, supporting its putative role as an intracellular messenger for Ca2+ release in human airway smooth muscle.