NIH3T3 cells stably expressing the rat 5-hydroxytryptamine 2A (5-HT 2A) receptor (5500 fmol/mg) were used to explore further the capacity of structurally distinct ligands to elicit differential signaling through the phospholipase C (PLC) or phospholipase A 2 (PLA 2) signal transduction pathways. Initial experiments were designed to verify that 5-HT 2A receptor-mediated PLA 2 activation in NIH3T3 cells is independent from, and not a subsequent result of, 5-HT 2A receptor-mediated PLC activation. In addition, we also explored the extent of receptor reserve for the endogenous ligand, 5-HT, for both PLC and PLA 2 activation. Finally, we employed structurally diverse ligands from the tryptamine, phenethylamine, and ergoline families of 5-HT 2A receptor agonists to test the hypothesis of agonist-directed trafficking of 5-HT 2A receptor-mediated PLC and PLA 2 activation. To measure agonist-induced pathway activation, we determined the potency and intrinsic activity of each compound to activate either the PLA 2 pathway or the PLC pathway. The results showed that a larger receptor reserve exists for 5-HT-induced PLA 2 activation than for 5-HT-induced PLC activation. Furthermore, the data support the hypothesis of agonist-directed trafficking in NIH3T3-5HT 2A cells because structurally distinct ligands were able to induce preferential activation of the PLC or PLA 2 signaling pathway. From these data we conclude that structurally distinct ligands can differentially regulate 5-HT 2A receptor signal transduction.