Previous work has shown that 5-hydroxytryptamine (5-HT)2A receptors can be radiolabelled with various radioligands, including partial agonists, such as [125I]-DOI and [3H]-DOB, and antagonists, such as [3H]-ketanserin and [3H]-spiperone. Because 5-HT has high affinity for the 5-HT2A receptor when displacing [3H]-DOB, the purpose of the present study was to determine whether or not the receptor could be labelled with [3H]-5-HT and what would be the effect of labelling the receptor with various radioligands having differing efficacies at the receptor. Consequently, the human 5-HT2A receptor stably expressed in NIH 3T3 cells was radiolabelled with the endogenous agonist [3H]-5-HT, the partial agonist [3H]-DOB, and the antagonist [3H]-ketanserin. The receptor could be radiolabelled with [3H]-5-HT with a Kd value of 1.3 +/- 0.1 nM and a Bmax value of 3461 +/- 186 fmoles/mg protein and the radiolabelling was sensitive to the stable guanosine 5'-triphosphate (GTP) analogue guanylyl-imidodiphosphate (GMP-PNP). Ketanserin labeled significantly more receptors (Kd = 1.1 +/- 0.1 nM: Bmax = 27,684 +/- 1500 fmoles/mg protein) than [3H]-DOB (Kd = 0.8 +/- 0.08 nM: Bmax = 8332 +/- 16 fmoles/mg protein) which, in turn, labelled significantly more receptors than [3H]-5-HT. The apparent affinity of antagonists did not change when the receptor was radiolabelled with either [3H]-agonists or [3H]-antagonists; however, agonists had a higher apparent affinity for [3H]-agonist-labeled receptors than for [3H]-antagonist-labeled receptors. Therefore, the apparent affinity of agonists for the 5-HT2A receptor estimated from displacement experiments depends on the intrinsic efficacy of the radioligand used.