Calcitonin gene-related peptide (CGRP) released from sensory neurons, which are closely apposed to mast cells and blood vessels, mediates gastric hyperemia in response to acid challenge of the damaged mucosa. Substance P (SP) is coreleased with CGRP from sensory neurons, but the role of this peptide in gastric blood flow regulation is largely unknown. Chambered rat stomachs were exposed to 1.5 M NaCl and acidic saline after treatment with SP, aprotinin (serine protease inhibitor), and the mast cell stabilizers ketotifen and sodium cromoglycate (SCG). Gastric hyperemia (measured with a laser Doppler flow velocimeter) after hypertonic injury and acid challenge was nearly abolished by SP. Aprotinin infused together with SP and pretreatment with ketotifen and SCG before SP restored the gastric hyperemia. Ketotifen and SCG inhibited mast cell degranulation in SP-treated rats. Preservation of gastric hyperemia was correlated with improved mucosal repair. These data suggest that impaired hyperemia by SP during acid challenge of the gastric mucosa may be mediated by a mast cell-dependent mechanism involving the release of proteases from mast cells.