Direct projections from the nucleus reuniens (Re) and the rhomboid nucleus (Rh) in the midline of the thalamus were examined in the rat by utilizing anterograde axonal transport of Phaseolus vulgaris leukoagglutinin (PHA-L). The Re projected axon terminals densely to the anterior cingulate cortex, infralimbic area of the medial frontal cortex, agranular insular cortex, entorhinal cortex, and parasubiculum. Labeled terminals in these cerebral cortices were distributed mainly in layer I, except for the entorhinal cortex where labeled terminals were distributed in layers I-IV. On the other hand, labeled terminals of projection fibers from the Rh to the cerebral cortex were distributed in layers II-VI of the anterior cingulate cortex, frontal cortex, parietal cortex, and agranular insular cortex. In the nucleus accumbens, caudate putamen, and basolateral amygdaloid nucleus, labeled terminals from the Rh were more dense than those from the Re. In the hypothalamus, labeled terminals from the Re were more dense than those from the Rh. The septal nuclei and field CA1 of Ammon's horn received labeled axon terminals from the rostral portion of the Re, but not from the caudal portion of the Re or the Rh. The patterns of distribution of axon terminals from the Re and Rh were similar in other subcortical target regions. To confirm the PHA-L results, wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) was injected into two projection sites of PHA-L-labeled axons. After injection into the agranular insular cortex, WGA-HRP-labeled neurons were found in the Re, but only a few were present in the Rh. In contrast to this, after WGA-HRP injection into the nucleus accumbens, WGA-HRP-labeled neurons were found in the Rh, but only a few were seen in the Re.