Ephrins and Eph receptors have been implicated to play important roles in axon guidance. A variable spacer region exists that differs significantly among distinct ephrins. An ephrin-A5 isoform has previously been isolated which lacks 27 amino acids within the spacer region. The expression and biological activities of this isoform, as well as the existence of isoforms for other ephrins that show variation within the spacer region, remain unknown. We report here a novel alternatively spliced isoform of ephrin-A3 which lacks the corresponding variable region. When compared to the longer isoforms, the shorter isoforms of both ephrin-A3 and ephrin-A5 remained less prominent in the brain during development, though their expression increased at postnatal stages. In addition, they could inhibit neurite outgrowth of dorsal root ganglia (DRG) neurons, suggesting that the corresponding variable regions were not essential for their axon guidance activities.