Although it is known that decreased ATP levels in liver and kidney contribute to organ dysfunction after shock, it remains unknown whether there is any association between decreased splenocyte ATP levels and splenic immune functions. Moreover, although studies have shown that ATP-MgCl2 treatment after shock improves hepatic and renal ATP levels and organ function, it remains unknown whether splenocyte ATP levels and immune functions are similarly affected by this agent. To study this, C3H/HeN mice were bled to a mean blood pressure of 35 mmHg, maintained at that pressure for 60 min, resuscitated with shed blood and Ringer lactate, and treated with ATP-MgCl2 (80 μmol/kg) or vehicle (saline). Splenocytes (SPL) were harvested at various intervals after hemorrhage, ATP levels were assessed by 31P nuclear magnetic resonance spectrometry, and functions were determined by measuring proliferative capacity and interleukin (IL)-2, and IL-3 synthesis. Hemorrhage depleted SPL ATP levels to 1.7 ±0.8% of control levels. However, there was a significant increase in ATP levels of ATP-MgCl2-treated mice (77 ± 11%, P < 0.05) compared with vehicle-treated animals (13 ± 4.1%) at 1 h after resuscitation. SPL ATP levels returned to control by 2 h after resuscitation in the ATP-MgCl2 group, whereas ATP levels of the vehicle-treated mice remained significantly depressed (P < 0.05) for up to 12 h after resuscitation. At 1 h after resuscitation, SPL proliferative capacity and IL-2 and IL-3 synthesis were all profoundly depressed in the vehicle-treated group (P < 0.05 vs. control). However, ATP-MgCl2 treatment significantly improved (P < 0.05) SPL proliferation and IL-2 and IL-3 synthesis, relative to vehicle-treated mice. These results indicate that 1) the markedly decreased SPL ATP may be the cause of early immunodepression after hemorrhage and 2) both SPL ATP levels and SPL immune functions can be restored with ATP-MgCl2 treatment after hemorrhage.