We have previously shown that subcutaneous bee venom (BV) injection reduces visceral pain behavior in mice, but it is not clear which constituent of BV is responsible for its antinociceptive effect. In the present study, we now demonstrate that a water-soluble subfraction of BV (BVA) reproduces the antinociceptive effect of BV in acetic acid-induced visceral pain model. We further evaluated three different BVA subfractions that were separated by molecular weight, and found that only the BVAF3 subfraction (a molecular weight of <10 kDa) produced a significant antinociceptive effect on abdominal stretches and suppressed visceral pain-induced spinal cord Fos expression. Injection of melittin (MEL), a major constituent of BVAF3, also produced a visceral antinociception. However, melittin's antinociception was completely blocked by boiling for 10 min at 100 degrees C, while boiling either whole BV or BVAF3 did not prevent their antinociception. The antinociceptive effect of BVAF3 was completely blocked by intrathecal pretreatment with the alpha2-adrenoceptor antagonist, yohimbine (YOH), while intrathecal pretreatment with the opioid antagonist, naloxone (NAL) or the serotonin antagonist, methysergide, had no effect. These data demonstrate that BVAF3 is responsible for the visceral antinociception of whole BV and further suggest that this effect is mediated in part by spinal alpha2-adrenergic activity.