Eph receptor tyrosine kinases have been implicated in various developmental processes, including axonal guidance, angiogenesis and morphogenesis. In general, Eph receptors and their ligands, ephrins, are reciprocally compartmentalized during embryogenesis. However, they are expressed in an overlapping fashion in some developing neural and non-neural tissues. Results from the overexpression or mutant mice of ephrin ligands in the retino-tectal system suggest that ephrin-As regulate co-expressed EphA receptor activity, but little is known about the molecular mechanisms. Here we show that EphA receptors and co-expressed ephrin-A ligands interact directly in cis via their functional binding domains, and that this interaction does not seem to mediate intracellular signals, but has an inhibitory effect on the trans interaction.