To determine the relative contributions of Ca²⁺ signaling and Ca²⁺ sensitivity to the contractility of airway smooth muscle cells (SMCs), we compared the contractile responses of mouse and rat airways with the lung slice technique. Airway contraction was measured by monitoring changes in airway lumen area with phase-contrast microscopy while changes in intracellular calcium concentration ([Ca²⁺]_i) of the SMCs were recorded with laser scanning microscopy. In mice and rats, methacholine (MCh) or serotonin (5-HT) induced concentration-dependent airway contraction and Ca²⁺ oscillations in the SMCs. However, rat airways demonstrated greater contraction as compared to mice, in response to agonists-induced Ca²⁺ oscillations of a similar frequency. Because this indicates that rat airway SMCs have a higher Ca²⁺ sensitivity compared to mice, we examined Ca²⁺ sensitivity with Ca²⁺-permeabilized airway SMCs in which the [Ca²⁺]_i was experimentally controlled. In the absence of agonists, high [Ca²⁺]_i induced a sustained contraction in rat airways but only a transient contraction in mouse airways. This sustained contraction of rat airways was relaxed by Y-23672, a Rho kinase inhibitor, but not affected by GF109203X, a protein kinase C inhibitor. The subsequent exposure of Ca²⁺-permeabilized airway SMCs, with high [Ca²⁺]_i, to MCh elicited a further contraction of rat airways and initiated a sustained contraction of mouse airways, without changing the [Ca²⁺]_i of the SMCs. Collectively, these results indicate that airway SMCs of rats have a substantially higher innate Ca²⁺ sensitivity than mice and that this strongly influences the transduction of the frequency of Ca²⁺ oscillations into the contractility of airway SMCs.