This study characterized mast cell- and capsaicin-sensitive sensory nerve vasodilator mechanisms regulating submucosal arterioles in the guinea pig ileum. The outside diameter of arterioles in in vitro submucosal preparations from milk-sensitized guinea pigs was monitored using videomicroscopy. Superfusion of the cow's milk protein, -lactoglobulin (-Lg; 5 µM), evoked large dilations, which became completely desensitized. -Lg-evoked dilations were blocked by pyrilamine or N^G-monomethyl-L-arginine plus indomethacin but not by TTX. Electron microscopic studies revealed that mast cells, in preparations receiving -Lg, demonstrated significant reductions of the dispersed and intact granule areas compared with preparations not exposed to -Lg. Paired experiments were conducted to determine if capsaicin-sensitive, nerve-evoked responses involved mast cell degranulation. One preparation received capsaicin (200 nM) followed by -Lg (5 µM); the other preparation received the drugs in reverse order. Prior treatment with capsaicin or -Lg had no effect on subsequent dilations evoked by the alternate treatment. Electron microscopy showed that nerve-arteriole associations were 10 times closer than nerve-mast cell associations. Mast cell numbers were not increased by milk sensitization. These findings suggest that mast cell- and capsaicin-sensitive nerve-evoked vasodilator mechanisms act independently in a model in which mast cell numbers are not increased.