Antipsychotic drugs are known to produce analgesic effects, but the mechanisms are not well established. In addition, some calmodulin inhibitors have been reported to produce antinociceptive effects. Among antipsychotic drugs, trifluoperazine is one of the most potent calmodulin inhibitors. We tried to find which of the known properties of this drug, including antidopaminergic, anticholinergic, antiadrenergic, antihistaminic, and calmodulin inhibition is involved in its analgesic effect. Various agents were injected through chronic subarachnoid catheter and their effects on formalin test were evaluated 15 minutes after drug administration. Since antinociceptive measures with formalin test are based on motor function, we also used rotarod test to quantify motor side effects of the drugs. Intrathecal trifluoperazine showed antinociceptive effects in a biphasic fashion i.e., analgesia at relatively low doses (1, 10µg/rat) and hyperalgesia at the higher dose (100µg/rat). No analgesic effect was observed after intrathecal injection of sulpiride, atropine, phentolamine and brompheniramine. Meanwhile, intrathecal calmidazolium induced a dose dependent analgesia (10,50,250 µg/rat). Low doses of histamine (1 µg/rat), physostigmine (1 µg/rat), bromocriptine (1 µg/rat) and norepinephrine (1 µg/rat) did not affect trifluoperazine induced analgesia. Calcium attenuated antinociceptive effect of trifluoperazine and inhibited analgesic effect of calmidazolium. Finally, naloxone decreased trifluoperazine induced antinociception but did not have any effect on calmidazolium-induced analgesia. We concluded that calmodulin inhibition may be involved in the analgesia produced by trifluoperazine. With increasing doses of trifluoperazine, algesic effect seems to overcome the analgesic effect. It is also suggested that the opioidergic system does not connect with "calmodulin inhibition"-induced analgesia eventhough this system has partial role in trifluoperazine-induced analgesia.