Regions of the prefrontal cortex that project to the nucleus accumbens in the rat receive input from midline thalamic and basal amygdaloid nuclei which also project to the same striatal region as their prefrontal cortical target. For example, the prelimbic cortex projects to the medial nucleus accumbens, and receives input from the paraventricular thalamic nucleus and the parvicellular basal amygdala. These latter two areas also project to the medial nucleus accumbens. It has been shown that afferents from the prelimbic cortex, the paraventricular thalamic nucleus and the parvicellular basal amygdala to the nucleus accumbens overlap or are separated in the nucleus accumbens, depending upon their position in the shell and core. The dorsal agranular insular cortex, the intermediodorsal thalamic nucleus and the magnocellular basal amygdaloid nucleus terminate in the lateral part of the nucleus accumbens and adjacent ventral part of the caudate-putamen. The intermediodorsal thalamic nucleus and the magnocellular basal amygdaloid nucleus reach both the dorsal agranular insular cortex and the lateral nucleus accumbens, and thus appear positioned to influence the prefrontal corticostriatal system at cortical and striatal levels. However, all three afferent systems have a heterogeneous distribution within this striatal region, and whether these projections actually reach the same areas is unknown. We investigated the patterns of separation and overlap in the nucleus accumbens between dorsal agranular insular cortical, magnocellular basal amygdaloid and intermediodorsal thalamic afferents with respect to the histochemical features of the nucleus. Techniques allowing the detection of two different anterograde tracers, or a single anterograde tracer and Calbindin-D28k immunoreactivity, in the same tissue sections were used. The results demonstrate that the afferents from the dorsal agranular insular area and the intermediodorsal thalamic nucleus avoid the shell of the lateral nucleus accumbens, which receives strong inputs from the magnocellular basal amygdala. In the matrix of the core and the ventral part of the caudate-putamen, fibers from the superficial layers of the dorsal agranular insular area overlap precisely with afferents from the intermediodorsal nucleus. In the patches, projections from the deep layers of the dorsal agranular insular cortex coincide with those from the magnocellular basal amygdala. The present findings have implications for the compartmental structure of the nucleus accumbens and provide novel insights into the organizational principles of prefrontal corticostriatal circuits.