We investigated the effect the loss of the CAT-2 gene (CAT-2-/-) has on lung resistance (R(L)) and tracheal isometric tension. The R(L) of CAT-2-/- mice at a maximal dose of acetylcholine (ACh) was decreased by 33.66% (P = 0.05, n = 8) compared with that of C57BL/6 (B6) mice. The isometric tension of tracheal rings from CAT-2-/- mice showed a significant decrease in carbachol (CCh)-induced force generation (33.01%, P < 0.05, n = 8) compared with controls. The isoproterenol- or the sodium nitroprusside-induced relaxation was not affected in tracheal rings from CAT-2-/- mice. The activity of iNOS and arginase in lung tissue lysates of CAT-2-/- mice was indistinguishable from that of B6 mice. Furthermore, the expression of phospholipase-Cbeta (PLC-beta) and phosphatidylinositol-(4)-phosphate-5-kinase-gamma (PIP-5K-gamma) was examined in the lung tissue of CAT-2-/- and B6 mice. The expression of PIP-5K-gamma but not PLC-beta was significantly reduced in CAT-2-/- compared with B6 mice. The reduced airway smooth muscle (ASM) contractility to CCh seen in the CAT-2-/- tracheal rings was completely reversed by pretreating the rings with 100 muM spermine. This increase in the CAT-2-/- tracheal ring contraction upon spermine pretreatment correlated with a recovery of the expression of PIP-5K-gamma. Our data indicates that CAT-2 exerts control over ASM force development through a spermine-dependent pathway that directly correlates with the expression level of PIP-5K-gamma in the lung.