Two novel mouse genes, Ebf2 and Ebf3, have been identified which show high similarity to the rodent Ebf/Olf-1 and the Drosophila collier genes. The strong conservation of the protein regions corresponding to the DNA binding and dimerisation domains previously defined in Ebf strongly suggests that Ebf2 and Ebf3 also constitute DNA sequence-specific transcription factors. Determination of the chromosomal locations of the two genes indicated that the different members of this novel mouse multigene family are not clustered. A detailed analysis of the expression of each of the three Ebf genes in the developing central nervous system revealed partially overlapping patterns with two salient features: 1) In the region extending from the midbrain to the spinal cord, the expression of the three genes correlated with neuronal maturation, with a general activation in early post-mitotic cells, followed by specific patterns of extinction also consistent with the neurogenic gradient. 2) In the forebrain area, although the patterns of expression of the Ebf genes also reflected neuronal maturation, they appeared in addition to be region specific. These data suggest that Ebf genes may be involved in the control of neuronal differentiation in the CNS and in enforcing regional diversity in populations of post-mitotic forebrain neurons. Dev. Dyn. 1997;210:191–205. © 1997 Wiley-Liss, Inc.