This study investigates the ontogeny of two transmitter systems which innervate the sexually dimorphic cremaster nucleus (CN). Since these transmitter systems arise from separate extra- and intraspinal sources, their ontological sequences differ. In males, substance P (SP) innervates CN motoneurons on postnatal day (P) 0, while serotonin (5-HT) is first observed on P2. SP reaches a mature innervation pattern on P20, while 5-HT maturation is prolonged to P40-60. It is suggested that part of the reason for the differing rates of development of 5-HT and SP within CN is due to the source of the innervating fibers; 5-HT descends from the brainstem while SP is of intraspinal origin. On P6, the SP innervation of the dorsal processes of the male CN is so advanced that the spinal cord may be 'sexed' according to the male presence, or the female 'absence', of this SP pattern. In developing and adult females, the 5-HT innervation of the CN is greater than the SP innervation. In addition, the 5-HT and SP innervation of the female CN is greater than the surrounding motor columns. Compared to surrounding ventral gray horn motor nuclei, the CN receives a slightly delayed SP and 5-HT innervation and this may be a reflection of the postnatal formation of the cremaster muscle.