In GP trachea that are precontracted with methacholine (MCh; 3H10'^-7 M), hyperosmolar D-mannitol (D-M) induces changes in epithelial Na+ and Clr transport and smooth muscle relaxation mediated by epitheliumderived relaxing factor (EpDRF). We examined the effects of sequentially-added ion channel blockers, MCh and D-M (30 mosM) on EC cell volume (CV) changes in ECs isolated enzymatically (2% protease, 1 h) from GP-trachea and suspended in Krebs-Henselejt solution, under conditions in which these agents are used in the isolated, perfused trachea preparation to examine D-M-induced EpDRF release. CV was measured with a cell sizer. D-M elicited shrinkage of EC (approximately 15 %) under conditions in which lit caused EpDRF-mediated relaxation. The Na+ channel blocker, amiloride (A; 10^-4 M), did not affect EC CV. MCh had no effect on CV (+/- A). Shrinkage stimulated by D-M was unaffected by A. On the other hand, incubation with the clr blocker, DIDS (10^-4 M), resulted in an immediate and prolonged decrease in CV (approximately 9%). Again, MCh had no effect on CV (+/- DIDS). However, in the presence of DIDS the CV decrease in response to D-M was diminished. Thus, the inhibitory effect of A on EpDRF release is not due to an effect on EC shrinkage, whereas the inhibitory effect of DIDS on EpDRF release may be linked to the decrease in, the CV response.