Although hyperosmolarity associated with diabetes is known to attenuate contractile response of airway smooth muscle, intracellular mechanisms involved are not fully understood. We examined the effects of hyperosmolarity on carbachol (CCh)- and aluminum fluoride (AF)-induced contractile and phosphatidylinositol (PI) responses of rat trachea. In vitro measurements of isometric tension and [³H] inositol monophosphate (IP₁) formed were conducted by using rat tracheal rings and slices. Hyperosmolarity solutions of 350, 450 and 600 mOsm were made with dissolving glucose in Krebs-Henseleit (K-H) solution. Hyperosmolarity attenuated dose-dependently CCh-induced contraction of rat trachea (1.86 ± 0.13 g at 300 mOsm, 1.85 ± 0.16 g at 350 mOsm, 1.37 ± 0.07 g at 450 mOsm and 0.50 ± 0.04 g at 600 mOsm, respectively), and also attenuated CCh- induced IP₁ accumulation (5.77 ± 0.33 Bq at 300 mOsm, 3.38 ± 0.26 Bq at 350 mOsm, 2.08 ± 0.30 Bq at 450 mOsm and 1.71 ± 0.40 Bq at 600 mOsm, respectively), and AF-induced IP₁ accumulation (3.93 ± 0.22 Bq at 300 mOsm, 1.63 ± 0.14 Bq at 450 mOsm and 1.02 ± 0.14 Bq at 600 mOsm, respectively). The results suggest that hyperosmolarity would inhibit G-protein-coupled phospholipase C, resulting in attenuation of CCh-induced airway smooth muscle contraction.