||Eph receptors belong to the largest family of receptor tyrosine kinases. Interactions with their ligands, known as ephrins, have been implicated to play important roles in axon guidance. Recent evidence indicates that ephrin-A5 is expressed in muscle during early development and participates in the topographic mapping of motor neurons during muscle innervation. An ephrin-A5 isoform has previously been isolated which lacks twenty-seven amino acids within the variable region near the carboxyl terminus. The expression and biological activities of this isoform, as well as the existence of isoform for other ephrins, remain unknown. In the present study, a novel alternatively spliced isoform of ephrin-A3, which lacked the corresponding variable region, was identified. The shorter isoforms of both ephrin-A3 and ephrin-A5 remained less prominent in the brain and muscle throughout development, but their expression increased during postnatal stages of the brain. In addition, they could inhibit neurite outgrowth of DRG neurons, suggesting that the corresponding variable regions were not essential for their axon guidance activities. While the participation of ephrins and Eph receptors in guiding motor axons during muscle innervation has been well documented, little is known about their expression and functional significance in muscle at later developmental stages. The present study investigates the expression and localization of Eph receptors and ephrins in skeletal muscle. Prominent expression of multiple Eph rceptors and ephrins was detected in muscle during embryonic development. More importantly, EphA4, A7, B3 and B4, as well as the ligand ephrin-A2, were localized at the neuromuscular junction (NMJ) of adult muscle. Despite their relative abundance, they were not localized at the synapses during embryonic stages. The concentration of EphA4, EphA7, and ephrin-A2 at the NMJ was observed at postnatal stages, and the synaptic localization became more prominent at later developmental stages. Expression of different Eph receptors in muscle was differentially regulated by neuregulin and after nerve injury. Furthermore, the present study demonstrated that over-expression of EphA4 led to tyrosine phosphorylation of the actin-binding protein cortactin, and that EphA4 was co-immunoprecipitated with cortactin in muscle. Taken together, these findings indicate that EphA4 is associated with the actin cytoskeleton. Since actin cytoskeleton is critical to the formation and stability of NMJ, the present findings raise the intriguing possibility that Eph receptors may have a novel role in NMJ formation and/or maintenance.