AbstractRetinal ganglion cells (RGCs) extend axons that exit the eye, cross the midline at the optic chiasm, and synapse on target cells in the optic tectum. Class three semaphorins (Sema3s) are a family of molecules known to direct axon growth. We undertook an expression screen to identify sema3s expressed in the retina and/or brain close to in‐growing RGC axons, which might therefore influence retinal‐tectal pathfinding. We find that sema3Aa, 3Fa, 3Ga, and 3Gb are expressed in the retina, although only sema3Fa is present during the time window when the axons extend. Also, we show that sema3Aa and sema3E are present near or at the optic chiasm. Furthermore, sema3C, 3Fa, 3Ga, and 3Gb are expressed in regions of the diencephalon near the path taken by RGC axons. Finally, the optic tectum expresses sema3Aa, 3Fa, 3Fb, and 3Gb. Thus, sema3s are spatiotemporally placed to influence RGC axon growth. Developmental Dynamics 236:2918–2924, 2007. © 2007 Wiley‐Liss, Inc.