Abstract In the spinal cord, motor neurons (MNs) with similar muscle targets and sensory inputs are grouped together into motor pools. To date, relatively little is known about the molecular mechanisms that control the establishment of pool-specific circuitry. Semaphorins, a large family of secreted and cell surface proteins, are important mediators of developmental processes such as axon guidance and cell migration. Here, we used mRNA in situ hybridization to study the expression patterns of semaphorins and their receptors, neuropilins and plexins, in the embryonic mouse spinal cord. Our data show that semaphorins and their receptors are differentially expressed in MNs that lie in distinct locations within the spinal cord. Furthermore, we report a combinatorial expression of class 3 (secreted) semaphorins and their receptors that characterizes distinct motor pools within the brachial and lumbar spinal cord. Finally, we found that a secreted semaphorin, Sema3A, elicits differential collapse responses in topologically distinct subpopulations of spinal MNs. These findings lead us to propose that semaphorins and their receptors might play important roles in the sorting of motor pools and the patterning of their afferent and efferent projections.