In the rat, fibers from the prelimbic cortex terminate in the medial nucleus accumbens. Anterior paraventricular thalamic and parvicellular basal amygdaloid fibers reached both the prelimbic cortex and the medial nucleus accumbens. All three afferent systems have an inhomogenous distribution within the nucleus accumbens, and whether or not these projections actually reach the same areas is unknown. Our aim was to evaluate the relationships of the three afferents with respect to the shell, the core, and the cell clusters of the nucleus accumbens. Double anterograde tracing and single anterograde tracing combined with immunohistochemistry for calbindin (D28k) or Nissl stain was used. Following tracer injections in the prelimbic cortex and the anterior paraventricular thalamus, a complementary (i.e., nonoverlapping) pattern of fibers was found in the shell. Thus, afferents from the prelimbic cortex are associated with cell clusters, whereas those from the anterior paraventricular thalamus avoid these cells but are affiliated with regions exhibiting weak homogeneous calbindin immunoreactivity. In the calbindin-poor patches of the core, the situation is reversed as both sets of fibers overlap. In cases with injections in the prelimbic cortex and the parvicellular basal amygdala, a pattern of overlap was seen in the shell and core. Thus, the fibers in the shell were found together in association with cell clusters, whereas regions of weak homogeneous calbindin immunoreactivity were avoided. In the core, overlap was seen in the patch compartment. Finally, with parvicellular basal amygdala/paraventricular thalamus injections, a complementary fiber organization was present in the shell, but overlap was prominent in the patches of the core. The results demonstrate that the relationships of prelimbic cortical, paraventricular thalamic, and parvicellular basal amygdaloid afferents in the nucleus accumbens vary according to their compartmental (immunohistochemical and cellular) affiliation. Compartmentalization is therefore a possible anatomical substrate for condensation or segregation of neuronal signals passing through the nucleus accumbens.