AbstractThe trigeminal ganglion provides the somatosensory innervation for the anterior rat tongue. At early embryonic stages (embryonic day [E] 12–13) pre‐tongue explants repel trigeminal axon outgrowth, and this is mediated by Sema3A (Rochlin and Farbman [1998] J. Neurosci. 18:6840–6852; Rochlin et al. [2000] J. Comp. Neurol. 422:579–593). Despite a decrease in repulsion by E14 and older tongue explants, Sema3A mRNA persists throughout the dorsal epithelium through E18, after axons have begun to penetrate papilla epithelium. We investigated the hypothesis that Sema3A continues to act as a repellent and that subpopulations of trigeminal axons that penetrate the epithelium become unresponsive to Sema3A. Sema3A repelled trigeminal axons in vitro regardless of the neurotrophic factor used to stimulate axon outgrowth, but the minimum level of Sema3A required to repel depended on the neurotrophic factor. Thus, in vitro, trigeminal axons are repelled by Sema3A when they would be penetrating the Sema3A‐mRNA rich epithelium in vivo. Whereas dorsal epithelium on tongue explants dissected at stages preceding target contact (E15) repelled trigeminal axons in vitro, explants dissected at later stages (E18), after axons would have penetrated the epithelium in vivo, were not repellent. To determine whether Sema3A prevents premature target penetration in vivo, we assessed the timing of target contact by sensory axons in Sema3A−/minus; and +/+ mice. Contact of the epithelium occurs prematurely in Sema3A−/minus; mice, but not penetration. Taken together, our data imply that Sema3A acts as a short‐range repellent that regulates the timing of target contact by trigeminal axons. J. Comp. Neurol. 470:13–24, 2004. © 2004 Wiley‐Liss, Inc.