Acetaminophen is one of the most widely used analgesic drugs. Although the mechanism of analgesic action of acetaminophen is still not known, the involvement of the central serotonin (5-hydroxytryptamine: 5-HT) system is one possibility. In the present study, we examined the antinociceptive effect of acute and chronic intraperitoneally (i.p.) administered acetaminophen by tail flick latency measurements in the rat. A significantly increased tail flick latency was observed in acute and 15-day acetaminophen-treated rats, but not in 30-day acetaminophen-treated rats, at a dose of 400 mg/kg/day. To investigate the plasticity of receptors at postsynaptic membrane, we conducted a series of experiments by radioligand binding method on frontal cortex and brainstem membrane. The technique involved radioligand binding with [phenyl-4-3H]spiperone and ketanserin for studying 5-HT(2A) receptor characteristics. A significant decrease in the maximum number of 5-HT(2A) binding sites (Bmax) was demonstrated in all treatment groups with acetaminophen 300 and 400 mg/kg on frontal cortex membrane, whereas the value of the dissociation equilibrium constant (Kd) remained unchanged. The down-regulation of 5-HT(2A) binding sites in frontal cortex was of a lesser magnitude after 30 days of treatment and the tail flick latency was as in the control animals. These results suggest that down-regulation of 5-HT(2A) receptor in response to 5-HT release is a major step in the mechanism underlying analgesia produced by this agent. On the contrary, chronic use of acetaminophen may result in 5-HT depletion, which in turn produces re-adaptation of postsynaptic 5-HT(2A) receptors. These data provide further evidence for a central 5-HT-dependent antinociceptive effect of acetaminophen.