We have used the chicken mim-1 gene as a model to study the mechanisms by which transcription factors gain initial access to their target sites in compacted chromatin. The expression of mim-1 is restricted to the myelomonocytic lineage of the hematopoietic system where it is regulated synergistically by the Myb and CCAAT/enhancer binding protein (C/EBP) factors. Myb and C/EBP cooperate at two distinct cis elements of mim-1, the promoter and a cell-type-specific enhancer, both of which are associated with DNase I hypersensitive sites in myelomonocytic cells but not in mim-1-nonexpressing cells. Previous work has shown that ectopic expression of Myb and C/EBP activates the endogenous mim-1 gene in a nonhematopoietic cell type (fibroblasts), where the gene is normally completely silent. Here, we investigated the molecular details of this finding and show that the activation of mim-1 occurs by two independent mechanisms. In the absence of Myb, C/EBP triggers the initial steps of chromatin opening at the mim-1 enhancer without inducing transcription of the gene. mim-1 transcription occurs only in the presence of Myb and is associated with chromatin opening at the promoter. Our work identifies a novel function for C/EBP in the initial steps of a localized chromatin opening at a specific, physiologically relevant target region.The significant changes in gene expression that occur during processes such as differentiation, proliferation, and apoptosis are often controlled at the transcriptional level. In eukaryotic organisms, the transcriptional activation of genes occurs in the chromatin context. The basic unit of chromatin is the nucleosome, which consists of an octameric complex of the core histones H2A, H2B, H3, and H4 and 146 base pairs of DNA wrapped around the protein core and connected by linker DNA. Further folding of nucleosomes through the binding of histone H1 leads to compact higher-order structures in which the DNA is not easily accessible for transcription factors or the transcriptional machinery. Access by the transcription machinery to DNA is facilitated by various chromatin remodeling enzymes that cause localized changes of the chromatin structure. These remodeling complexes are themselves recruited by transcription factors to specific cis-acting sequences (for reviews see references 4, 23, 26, 41, 47, and 48). Although we have a rather detailed picture of the chromatin remodeling events that occur after transcription factors have associated with their binding sites, the mechanisms by which these proteins gain initial access to their binding sites in nucleosomally organized and tightly packed DNA are less well understood. Cirillo et al. (14) have used the term "pioneer factor" to describe a class of transcription factors that are capable of entering into silent chromatin and initiating chromatin opening.