The serotonin 2A (5-HT2A) receptor is necessary for the psychopharmacological actions of the serotonergic hallucinogens such as LSD. An exploration of the biophysical actions of hallucinogens at the 5-HT2A receptor may be useful in understanding their unique psychological effects, particularly in the elucidation of structure-activity relationships for developing potent receptor- and functionally-selective 5-HT2A agonists. Experiments were undertaken to optimize, validate, and explore the utility of an in silico-activated human 5-HT2A receptor homology model developed previously in our laboratory. In the original model, a number of receptor-ligand interactions were observed. The lack of strong empirical support for several of the interactions indicated in the original modeling provided opportunities to explore further the topology of the 5-HT2A receptor binding site, which also provides support for the model itself. The first section of this work describes a qualitative use of our h5-HT2A receptor homology model to provide a molecular basis for the pharmacological characterization of psychoactive phenylalkylamine hallucinogens. Subsequent sections detail a systematic iterative approach to explore several of the receptor-binding interactions observed in virtual docking simulations to our h5-HT2A receptor model. Data were generated by site-directed mutagenesis of h5-HT2A receptor residues, with binding and functional assays. Mutation of Phe6.51(339) and Phe6.52(340) to leucine residues gave results consistent with previous studies that indicated an aromatic interaction between Phe6.52(340) and 5-HT2A receptor agonists. Importantly, a novel role for Phe6.51(339) was identified, where it was found to interact with a new class of 5-HT2A receptor agonists. Data from the mutation of Gly5.42(238), Ser5.43(239), and Ser5.46(242) to alanine residues are consistent with the orientations of phenylalkylamines, tryptamines, and ergolines observed in the original development of our h5-HT2A receptor model. Mutation of Ser3.36(159) and Thr3.37(160) to alanine residues did not, however, provide data to support the hypothesis of hydrogen bond interactions between these residues and the 2-methoxy of phenylalkylamines. Similarly, data from the mutation of Asn6.55(343) failed to support the hypothesis of its interaction with the carbonyl of ergolines. Overall, the data from this work provide strong evidence to support the topology of our h5-HT2A receptor homology model, although further refinement of the model remains necessary.