The nucleus accumbens in the rat has been parcelled into shell and core subdivisions. Despite accumulating evidence for such a division of the nucleus accumbens, these territories have not been delineated throughout the rostrocaudal extent of the nucleus. In the present study, an attempt has been made to delineate the shell and core using the distribution of calcium-binding protein immunoreactivity, substance P immunoreactivity and acetylcholinesterase activity in transverse and horizontal sections through the nucleus accumbens. It was found that the pattern of calcium-binding protein immunoreactivity provides the most unequivocal criterion to divide the nucleus accumbens into a ventral and medial, peripheral shell displaying low to moderate immunostaining, and a more laterally and dorsally located, strongly stained inner core. In most parts of the nucleus, borders seen in the calcium-binding protein immunoreactivity pattern can also be recognized in the distributions of substance P immunoreactivity and acetylcholinesterase activity. It is concluded that the shell occupies most of the rostral part of the nucleus accumbens, whereas rostrally the core is represented only in the most lateral part. Differences in staining intensities for all three markers indicate that both the shell and core have a heterogeneous structure. Patterns of connectivity appear to support the division of the nucleus accumbens as indicated by calcium-binding protein immunoreactivity in the present study.