Semaphorins comprise a family of molecules that influence the growth and guidance of neuronal processes. Class-3 semaphorins are secreted proteins, and their effects are mediated by neuropilin (NP) and plexin (Plx) receptors. There is considerable information on mechanisms that influence axonal guidance and plasticity in the mammalian visual system, but a role for semaphorins has received less scrutiny. The purpose of the current study was to survey class-3 semaphorin and cognate receptor expression in young and adult rat retinas. The mRNA expression of five class-3 semaphorins (3A, 3B, 3C, 3E, and 3F) and receptor subtypes NP-1 and -2 and plexins A1 and A2 was determined, by using riboprobes and in situ hybridization on cryosections of newborn (postnatal day [P]0), juvenile (P14), and adult rat retinas. Retinal ganglion cells (RGCs) were identified by retrograde labeling after injections of a fluorescent tracer (Fluorogold, FG) into the superior colliculus. Hybridized sections were also immunostained to identify specific retinal cell classes. mRNA expression for all five members of the class-3 semaphorin family was seen in adult FG-labeled RGCs. Qualitatively, expression was highest for semaphorins 3B and 3C, and lowest for 3A. Levels of mRNA expression in RGCs were lower in newborn retinas but were adult-like by P14. Expression by different cell types in the inner nuclear layer was also seen, especially at P14. Expression of NP-2 and PlxA2 mRNAs was evident in developing inner nuclear and ganglion cell layers at birth. Expression increased postnatally and was maintained into adulthood. NP-1 and PlxA1 expression was also present, but at comparatively lower levels. The presence of class-3 semaphorins and their receptors in neonatal and adult rat retina suggests a potential role for these proteins in retinal development and in the maturation, stabilization, and plasticity of mammalian primary visual pathways.