Serotonin (5-hydroxytryptamine; 5-HT) and 5-HT2receptors belong to the class of phosphoinositide-specific phospholipase C (PLC)-linked receptors. Conditions were established for measuring 5-HT2A-linked and 5-HT2~-linkedPLC activity in membranes prepared from previously frozen rat frontal cortex and caudate. In the presence of Ca 2~(300 nM) and GTPyS (1 ftM), 5-HT increased PLC activity in caudate membranes. Pharmacological analysis using the selective 5-HT 2A antagonist, spiperone, and the nonselective 5-HT2AI2C antagonist, mianserin, demonstrated that over half of the 5-HT-stimulated PLC activity was due to stimulation of 5-HT2~receptors as opposed to 5-HT2A receptors. Radioligand binding assays with [ 3H]RP62203 and [3H]-mesulergine were used to quantify 5-HT 2A and 5-HT2s ites, respectively, in caudate. From these data, the Bmax for caudate 5-HT2A sites and 5-HT2~sites was 165.4 ii 9.7 fmol/mg of protein and 49.7 ±3.3 fmol/mg of protein, respectively. In contrast to that in caudate, PLC activity in frontal cortex was stimulated by 5-HT in a manner that was inhibited by the 5-HT2A-selective antagonists, spiperone and ketanserin. Taken together, the results indicate that 5-HT2A-and 5-HT2~-linkedPLC activity can be discerned in brain regions possessing both receptor subtypes using membranes prepared from previously frozen tissue. More importantly, significant 5-HT2~-mediated phosphoinositide hydrolysis was observed in caudate, despite the relatively low density of 5-HT2~sites. The significance of these observations with respect to the physiological function of 5-HT2~receptors is discussed. Key Words: Serotonin receptor-Phospholipase C-Phosphoinositide hydrolysis-Brain-Rat.