Rationale: The etiology of airway hyperresponsiveness associated with asthma requires an understanding of the regulatory mechanisms mediating human airway smooth muscle cell (SMC) contraction. Objectives: To determine how human airway SMC contraction (induced by histamine) and relaxation (induced by formoterol) are regulated by Ca2+ oscillations and Ca2+ sensitivity. Methods: 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. Ca2+ signaling and Ca2+ sensitivity of airway SMCs were measured with 2-photon fluorescence microscopy and Ca2+-permeabilized lung slices. Results: The agonist histamine induced contraction of human small airways by stimulating both an increase in [Ca2+]i in the SMCs in the form of oscillatory Ca2+ waves and an increase in Ca2+ sensitivity. The frequency of the Ca2+ oscillations increased with histamine concentration and correlated with increased contraction. Formoterol induced airway relaxation, at low concentrations, by initially decreasing SMC Ca2+ sensitivity. At higher concentrations, formoterol additionally slowed or inhibited the Ca2+ oscillations of the SMCs to relax the airways. The action of formoterol was only slowly reversed. Conclusions: Human lung slices provide a powerful experimental assay for the investigation of small airway physiology and pharmacology. Histamine induces contraction by simultaneously increasing SMC Ca2+ signaling and Ca2+ sensitivity. Formoterol induces long-lasting relaxation by initially reducing the Ca2+ sensitivity and, subsequently, the frequency of the Ca2+ oscillations of the airway SMCs.