Although diltiazem improves immune responses after hemorrhage and resuscitation, its mechanism remains unknown. To study this, C3H/HeN mice were bled to a mean blood pressure of 35 mmHg, maintained at that level for 1 h, and then adequately resuscitated and treated with diltiazem (400 μg/kg body wt) or saline (vehicle). One hour after hemorrhage and resuscitation, splenic microvascular blood flow was determined by laser Doppler flowmetry. Splenocytes were also harvested and ATP levels and cytoplasmic free Ca2+ concentration ([Ca2+](i)) were determined by 31P nuclear magnetic resonance and fluo 3 flow cytometry, respectively. Splenocyte functions were determined by measuring interleukin (IL)-1, IL-2, IL-3, IL-6, and tumor necrosis factor concentrations in concanavalin A-stimulated supernatant with cytokine-specific cellular assays. Hemorrhage and resuscitation caused a significant decrease in the splenocyte's ability to release cytokines, which was correlated with significant reductions in splenocyte ATP levels and splenic microvascular blood flow as well as a significant increase in splenocyte [Ca2+](i). Diltiazem significantly decreased splenocyte [Ca2+](i) while improving splenocyte ATP levels, cytokine production, and splenic microvascular blood flow. Nitroglycerin (71 μg/kg body wt) administration improved splenic microvascular blood flow to diltiazem- treated levels but failed to concomitantly improve splenocyte ATP levels or cytokine production. Thus diltiazem's immunoprotective effects appear to be the result of decreased Ca2+-induced splenocyte injury.