The purpose of the present investigations was (1) to examine the spatial organization of preganglionic neurons of the sacral parasympathetic nucleus in the lumbosacral spinal cord of male adult rats and (2) to search, in this nucleus, for a possible segregation of sub-populations of neurons innervating the penis or the bladder, respectively. To estimate their spatial organization, neurons of the sacral parasympathetic nucleus were retrogradely labeled by wheat germ agglutinin coupled to horseradish peroxidase applied to the central end of the sectioned pelvic nerve. The sub-populations of lumbosacral neurons innervating the corpus cavernosum of the penis or the dome of the bladder were identified using transsynaptic retrograde labeling by pseudorabies virus injected into these organs in different rats. In both wheat germ agglutinin-labeled and pseudorabies virus-labeled rats, serial coronal sections were cut through the spinal L5-S1 segments. Labeled neurons were revealed by histochemistry (peroxidase experiments) or immunohistochemistry (pseudorabies virus experiments). By means of a three-dimensional reconstruction software developed in our laboratory, three-dimensional models were calculated from each spinal section image series. They revealed the spatial organization of (i) preganglionic neurons and (ii) neurons innervating the bladder or the penis. The different three-dimensional models were subsequently merged into a single one which revealed the segregation, within the sacral parasympathetic nucleus, of the sub-populations of neurons. Neurons labeled by virus injected into the penis extended predominantly from the rostral part of the L6 segment to the rostral part of the S1 segment while those labeled by bladder injections were distributed predominantly from the caudal part of the L6 segment to the caudal part of the S1 segment. These results support the hypothesis of a viscerotopic organization of sacral neurons providing the spinal control of pelvic organs.