Intercellular signaling molecules of the vertebrate hedgehog family and transcription factors of the winged-helix family have been implicated in floor plate development. We have examined the consequences of misexpressing the vertebrate hedgehog gene vhh-1 (sonic hedgehog, shh) and the winged-helix gene HNF-3 beta in the neural plate and neural tube of frog embryos. Misexpression of either of these genes induces floor plate differentiation at ectopic locations. However, ectopic floor plate induction in response to both vhh-1 and HNF-3 beta was temporally and spatially restricted. At neural plate stages, ectopic floor plate differentiation was not detected. After neural tube closure, ectopic floor plate differentiation was detected but was restricted predominantly to the dorsal region of the neural tube. The ability of winged-helix and vertebrate hedgehog genes to induce floor plate differentiation in vivo may, therefore, be constrained by additional signals that specify the time and position of floor plate differentiation.