While the role of the notochord and floor plate in patterning the dorsal-ventral (D/V) axis of the neural tube is clearly established, relatively little is known about the earliest stages of D/V regionalization. In an effort to examine more closely the initial, preneural plate stages of regionalization along the prospective D/V neural axis, we have performed a series of explant experiments employing xHB9, a novel marker of the motor neuron region in Xenopus. Using tissue recombinants and Keller explants we show that direct mesodermal contact is both necessary and sufficient for the initial induction of xHB9 in the motor neuron region. We also show that presumptive neural plate explants removed as early as midgastrulation and cultured in isolation are already specified to express xHB9 but do so in an inappropriate spatial pattern while identical explants are specified to express the floor plate marker vhh-1 with correct spatial patterning. Our data suggest that, in addition to floor plate signaling, continued interactions with the underlying mesoderm through neural tube stages are essential for proper spatial patterning of the motor neuron region.