The 5-HT(2A) and 5-HT(2C) receptors belong to the same subtype of the G-protein coupled receptor family and have several agonist and antagonist ligands in common. To gain more insight into the differences in the regulation of the two receptors, we studied the effect of agonist and antagonist pre-treatment on radioligand receptor binding and 5-HT-induced inositol phosphate formation on rat 5-HT(2A) and rat 5-HT(2C) receptors stable expressed in NIH 3T3 cells. We compared short (15 min) and prolonged (48 h) pre-treatment of the cells with the natural agonist, 5-HT and with the antagonist pipamperone, which can be readily washed out. The rat 5-HT(2C) receptor showed an agonist-induced down-regulation (decrease in B(max) of labelled agonist and antagonist binding) and desensitisation (decrease in 5-HT-induced inositol phosphate formation and potency of 5-HT). Antagonist pre-treatment induced an increase in rat 5-HT(2C) receptor-mediated inositol phosphate formation as well as increased agonist and antagonist radioligand binding. These findings are consistent with the classical model of G-protein coupled receptor regulation. In contrast, the rat 5-HT(2A) receptor expressed in the same host cell behaved differently, unlike the classical model. Pre-treatment with 5-HT for 15 min and 48 h did not change receptor levels measured by radioligand binding, but the signal transduction response (inositol phosphate formation) was significantly reduced. Pre-treatment with the antagonist pipamperone for 15 min and 48 h caused an increase in antagonist radioligand binding but a reduction in agonist radioligand binding and a decrease in inositol phosphate formation and potency of 5-HT. Hence, the rat 5-HT(2A) receptor apparently undergoes agonist desensitisation without down-regulation of the total receptor number. Antagonist pre-treatment causes a paradoxical desensitisation, possibly by uncoupling of the receptor from G-proteins. The uncoupled receptor does not bind 5-HT in the nanomolar range but retains its antagonist binding properties. Paradoxical antagonist-induced desensitisation of rat 5-HT(2A) receptors has also been observed in vivo.