The role of glutamate as a neurotransmitter in the zebrafish olfactory bulb (OB) was established by examining neuronal activation following 1) glutamate receptor agonist stimulation of isolated olfactory bulbs and 2) odorant stimulation of intact fish. Four groups of neurons (mitral cells, projection neurons; granule cells, juxtaglomerular cells, and tyrosine hydroxylase-containing cells; interneurons) were identified on the basis of cell size, cell location, ionotropic glutamate receptor (iGluR) agonist/odorant sensitivity, and glutamate, γ-aminobutyric acid (GABA), and tyrosine hydroxylase immunoreactivity. Immunoreactive glutamate levels were highest in olfactory sensory neurons (OSNs) and mitral cells, the putative glutamatergic neurons. The sensitivity of bulbar neurons to iGluR agonists and odorants was established using a cationic channel permeant probe, agmatine (AGB). Agmatine that permeated agonist- or odor-activated iGluRs was fixed in place with glutaraldehyde and detected immunohistochemically. N-methyl-D-aspartic acid (NMDA) and α-amino-3-hydroxyl-5-methylisoxazole-4-propionic acid (AMPA)/kainic acid (KA) iGluR agonists and odorants (glutamine, taurocholic acid) stimulated activity-dependent labeling of bulbar neurons, which was blocked with a mixture of the iGluR antagonists, D-2-amino-5-phosphono-valeric acid (APV) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). The AMPA/KA antagonist CNQX completely blocked glutamine-stimulated AGB labeling of granule cells and tyrosine hydroxylase-containing cells, suggesting that, in these cell types, AMPA/KA receptor activation is essential for NMDA receptor activation. However, blocking AMPA/KA receptor activity failed to eliminate AGB labeling of mitral cells or juxtaglomerular cells. Collectively, these findings indicate that glutamate is the primary excitatory neurotransmitter in the zebrafish OB and that iGluR subtypes function heterogeneously in the bulbar neurons. J. Comp. Neurol. 454:294–309, 2002. © 2002 Wiley-Liss, Inc.