A number of gustatory receptors have been proposed to underlie umami, the taste of l-glutamate, and certain other amino acids and nucleotides. However, the response profiles of these cloned receptors have not been validated against responses recorded from taste receptor cells that are the native detectors of umami taste. We investigated umami taste responses in mouse circumvallate taste buds in an intact slice preparation, using confocal calcium imaging. Approximately 5% of taste cells selectively responded to l-glutamate when it was focally applied to the apical chemosensitive tips of receptor cells. The concentration–response range for l-glutamate fell approximately within the physiologically relevant range for taste behavior in mice, namely 10 mm and above. Inosine monophosphate enhanced taste cell responses to l-glutamate, a characteristic feature of umami taste. Using pharmacological agents, ion substitution, and immunostaining, we showed that intracellular pathways downstream of receptor activation involve phospholipase C β2. Each of the above features matches those predicted by studies of cloned and expressed receptors. However, the ligand specificity of each of the proposed umami receptors [taste metabotropic glutamate receptor 4, truncated metabotropic glutamate receptor 1, or taste receptor 1 (T1R1) and T1R3 dimers], taken alone, did not appear to explain the taste responses observed in mouse taste cells. Furthermore, umami responses were still observed in mutant mice lacking T1R3. A full explanation of umami taste transduction may involve novel combinations of the proposed receptors and/or as-yet-undiscovered taste receptors.