The etiology of airway hyperresponsiveness associated with asthma requires an understanding of the regulatory mechanisms mediating human airway smooth muscle cell (SMC) contraction. The objective of this study was to determine how human airway SMC contraction (induced by histamine) and relaxation (induced by formoterol) are regulated by Ca(2+) oscillations and Ca(2+) sensitivity. The responses of human small airways and their associated SMCs were studied in human lung slices cut from agarose-inflated lungs. Airway contraction was measured with phase-contrast video microscopy. Ca(2+) signaling and Ca(2+) sensitivity of airway SMCs were measured with two-photon fluorescence microscopy and Ca(2+)-permeabilized lung slices. The agonist histamine induced contraction of human small airways by stimulating both an increase in intracellular Ca(2+) concentration in the SMCs in the form of oscillatory Ca(2+) waves and an increase in Ca(2+) sensitivity. The frequency of the Ca(2+) oscillations increased with histamine concentration, and correlated with increased contraction. Formoterol induced airway relaxation at low concentrations by initially decreasing SMC Ca(2+) sensitivity. At higher concentrations, formoterol additionally slowed or inhibited the Ca(2+) oscillations of the SMCs to relax the airways. The action of formoterol was only slowly reversed. Human lung slices provide a powerful experimental assay for the investigation of small airway physiology and pharmacology. Histamine induces contraction by simultaneously increasing SMC Ca(2+) signaling and Ca(2+) sensitivity. Formoterol induces long-lasting relaxation by initially reducing the Ca(2+) sensitivity and, subsequently, the frequency of the Ca(2+) oscillations of the airway SMCs.