Vesicular glutamate transporter 1 (VGluT1) is one of the best markers for glutamatergic neurons, because it accumulates transmitter glutamate into synaptic vesicles. Differentiation‐associated Na⁺‐dependent inorganic phosphate cotransporter (DNPI) shows 82% amino acid identity to VGluT1, and is another candidate for vesicular glutamate transporters. Here, we report the immunocytochemical localization of DNPI and compare it with that of VGluT1 in the adult rat brain. Both DNPI and VGluT1 immunoreactivities were found mostly in neuropil, presumably in axon terminals, throughout the brain. In the telencephalic regions, intense DNPI immunoreactivity was observed in the glomeruli of the olfactory bulb, layer IV of the neocortex, granular layer of the dentate gyrus, presubiculum, and postsubiculum. In contrast, VGluT1 immunoreactivity was intense in the olfactory tubercle, layers I‐III of the neocortex, piriform cortex, entorhinal cortex, hippocampus, dentate gyrus, and subiculum. In the thalamic nuclei, DNPI‐immunoreactive terminal‐like profiles were much larger than VGluT1‐immunoreactive ones, suggesting that DNPI immunoreactivity was subcortical in origin. DNPI immunoreactivity was much more intense than VGluT1 immunoreactivity in many brainstem and spinal cord regions, except the pontine nuclei, interpeduncular nucleus, cochlear nuclei, and external cuneate nucleus. In the molecular layer of the cerebellar cortex, climbing‐like fibers showed intense DNPI immunoreactivity, whereas neuropil contained dense VGluT1‐immnoreactive deposits. Both DNPI and VGluT1 immunoreactivities were observed as mossy fiber terminal‐like profiles in the cerebellar granular layer. DNPI and VGluT1 immunoreactivities appeared associated with synaptic vesicles in the axon terminals forming asymmetric synapses in several regions examined electron microscopically. The present results indicate that DNPI and VGluT1 are used by different neural components in most, if not all, brain regions, suggesting the complementary functions of DNPI and VGluT1. J. Comp. Neurol. 444:39–62, 2002. © 2002 Wiley‐Liss, Inc.