We have cloned the human ionotropic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GluR3 flip splice variant (hGluR3i) and developed a stable cell line expressing this receptor in HEK293 cells. Electrophysiological recordings demonstrated that glutamate-evoked currents desensitize rapidly, with a mean desensitization time constant of 5.4 ms. Robust glutamate-evoked increases in intracellular Ca++ ([Ca++]i) were observed in the presence of cyclothiazide, which attenuated receptor desensitization. [Ca++]i measurements were used to perform a detailed pharmacological characterization of hGluR3i with reference agonists and antagonists. The results of these studies showed that kainate and domoate were not fully efficacious agonists relative to glutamate. The binding affinities of agonists and competitive antagonists were determined in a [3H]AMPA competition binding assay. There was a good correlation between the functional data and the binding affinities obtained for competitive antagonists. However, the binding affinities of the agonists did not correlate with their functional EC50 values from [Ca++]i data, possibly because the binding assay predominantly measures the desensitized high-affinity state of the receptor. [3H]AMPA binding also was performed on membranes prepared from rat forebrain, and comparison of the data from HEK293 cells expressing hGluR3i and rat forebrain suggest that nearly all of the reference compounds show similar binding activities between the two membrane preparations, with the exception of fluoro-willardiine, kainate and 6-nitro-7-sulfamoylbenzo(f)quinoxaline-2-3-dione (NBQX). These data suggest that cells stably expressing recombinant hGluR3i represent pharmacologically valid experimental systems to study human AMPA receptors.