Notochord has been implicated in previous studies in both the dorsoventral and rostrocaudal patterning of the developing neural tube. This possibility has been further explored by analyzing the expression of Engrailed-2 in chick embryos developing with cranial notochord defects. Control embryos containing intact notochords expressed Engrailed-2 protein within the neural tube and in a subset of the neural crest and overlying surface ectoderm at the future mesencephalon and cranial metencephalon levels. Within the neural tube, expression was confined to cell nuclei in the roof plate and lateral walls; floor plate nuclei directly overlying the notochord typically failed to show expression. After surgical removal of Hensen's node, the source of notochord precursor cells, embryos were cultured through neurulation and assayed for expression of Engrailed-2 protein. All embryos that partially or completely lacked cranial notochord expressed Engrailed-2 in a pattern similar to that of control embryos containing intact notochords, except that when notochord and floor plate were absent, Engrailed-2 was also expressed in the most ventral part of the neural tube. These results indicate that 1) Engrailed-2 expression is suppressed in the most ventral neural tube owing to induction of the floor plate by the notochord, and 2) that the presence of an underlying notochord is not required for correct rostrocaudal expression, suggesting that multiple pathways act in the patterning of the rudiment of the central nervous system.