Members of the C/EBP family of transcriptional factors have been implicated in the regulation of genes in a variety of tissues. We report here the isolation and characterization of the human C/EBPϵ gene (CEBPE). By using low-stringency hybridization conditions and probes derived from the C/EBPα and C/EBPδ genes, we have isolated overlapping genomic clones that cover almost 25 kb of the C/EBPϵ gene locus and corresponding cDNA clones. DNA sequence analysis reveals that the gene encodes a protein highly homologous to rat CRP1. The gene was assigned to chromosome 14q11.2 by fluorescencein situhybridization and was physically linked to the genetic marker D14S990. Based on linkage data derived from this marker, we positioned the CEBPE gene between the T-cell receptor α/δ locus and a cluster of four serine proteases expressed exclusively in hematopoietic cells. Expression of C/EBPϵ was detected in Jurkat T-cell and in HL 60 promyelocytic cell lines. From a variety of normal human tissues studied, expression of mRNA was monitored only in peripheral blood mononuclear cells, tissues involved in the immune system, and ovaries. These data demonstrate that the C/EBPϵ gene shows a restricted pattern of expression, has an intriguing chromosomal location, and suggest a possible role for the regulation of certain genes in cells of myeloid and lymphoid lineages.