The segmental organization of midthoracic rat and chicken sympathetic preganglionic neurons was examined by retrograde labeling in vivo and in vitro. The results demonstrate that individual sympathetic preganglionic neurons project only rostrally or caudally within the sympathetic chain, even though the spinal segment in which they reside provides innervation to both rostral and caudal ganglia. In addition, there is both a segmental and an intrasegmental pattern in the thoracic sympathetic column, in which the position of preganglionic neurons is related to the direction they project in the sympathetic chain. Thoracic sympathetic preganglionic neurons are organized into discrete segmental units, each of which exhibits an internal rostrocaudal polarity with respect to the direction of axon projection in the sympathetic chain. The rostrocaudal bias of this internal polarity is graded from segment to segment along the longitudinal axis. Since there is minimal overlap between thoracic segments, the transition from one segment to another entails a sharp change in the pathway choice of the preganglionic neurons. The organization of the preganglionic projections thus includes (1) segmental periodicity, (2) intrasegmental gradients of neuronal identity, and (3) an axial gradient of segment identity. The significance of these findings is twofold. First, they suggest a functional organization that may be related to the specificity of sympathetic reflex action. Second, they reveal a cellular organization that suggests an underlying segmental pattern in the developing spinal cord.