We have compared the expression of the murine genes En-1,En-2, and in-1 during development by in situ hybridization. Expression of all three genes was first detected at 8.0 days in overlapping bands of the anterior neural folds. By 12.0 days the expression patterns diverged. En-1 and En-2 were expressed in a similar ring of cells in the central nervous system (CNS) at the midbrain/hindbrain junction. En-1 was also expressed de novo in two lateral stripes extending the length of the hindbrain and spinal cord, in the developing vertebral column, in two lateral stripes of dermatome-derived cells, and in the tail and limb buds. By 12.0 days int-1 expression showed little overlap with the En genes and could not be detected at later stages. At 15.5 days En gene expression was primarily limited to the midbrain/hindbrain in overlapping but nonidentical sets of differentiated cells. In the adult, En-1 and En-2 marked the same sets of cells in the pons, but En-2 alone was detected in the granular layer of the cerebellum. The results are consistent with int-1 and the En genes playing a role early in development in defining spatial domains in the CNS. Later in development the En genes may have an additional function in neurogenesis. En-1 expression in the developing pericordal tube suggests that it may also be involved in vertebral assembly.