During Xenopus gastrulation, the internalizing mesendodermal cell mass is brought into contact with the multilayered blastocoel roof. The two tissues do not fuse, but remain separated by the cleft of Brachet. This maintenance of a stable interface is a precondition for the movement of the two tissues past each other. We show that separation behavior, i.e., the property of internalized cells to remain on the surface of the blastocoel roof substratum, spreads before and during gastrulation from the vegetal endoderm into the anterior and eventually the posterior mesoderm, roughly in parallel to internalization movement. Correspondingly, the blastocoel roof develops differential repulsion behavior, i.e., the ability to specifically repell cells showing separation behavior. From the effects of overexpressing wild-type or dominant negative XB/U or EP/C cadherins we conclude that separation behavior may require modulation of cadherin function. Further, we show that the paired-class homeodomain transcription factors Mix.1 and gsc are involved in the control of separation behavior in the anterior mesoderm. We present evidence that in this function, Mix.1 and gsc may cooperate to repress transcription.