Neomycin (3 mM) inhibited maximal 5-HT-induced contraction by approximately 50% without inhibiting [3H]5-HT binding to 5-HT2A receptors. In contrast, neomycin had no effect on carbamylcholine- or histamine-induced contraction. Carbamylcholine (10 microM) and histamine (10 microM) both stimulated phosphatidylinositol (PI) hydrolysis but neomycin had no effect on the increase in PI hydrolysis. 5-HT (10 microM) did not stimulate PI hydrolysis in the absence or presence of neomycin, suggesting that neomycin inhibited 5-HT contraction in the guinea pig trachea independent of PI turnover. Although bradykinin stimulated phospholipase D (PLD) activity, 5-HT did not activate PLD, suggesting that the 5-HT2A receptor is not coupled to this enzyme in the guinea pig trachea. Neomycin (3 mM) and nitrendipine (1 microM) inhibited 5-HT-induced contraction to a similar extent, and neomycin did not further inhibit contraction in the presence of nitrendipine. These data indicate that neomycin inhibited 5-HT-induced contraction, like nitrendipine, via an effect on calcium influx through L-type calcium channels and did not affect intracellular calcium release. However, unlike nitrendipine which completely blocked KCl-induced contraction, neomycin only marginally reduced the maximal KCl-induced contraction. Taken together, these data suggest that neomycin may indirectly inhibit calcium influx through L-type calcium channels in guinea pig tracheal smooth muscle. The mechanism by which neomycin inhibited calcium influx in the guinea pig trachea may provide insight into the novel signaling pathway of the 5-HT2A receptor in this tissue.