We recently cloned human and murine cDNAs that encode CP2, a transcription factor that interacts with the murine alpha-globin promoter. In this report, we exploited our ability to express CP2 in bacteria and eukaryotic cells to further investigate factor activities in vitro and in vivo. CP2 expressed in bacteria was significantly enriched and used in a series of DNase I footprinting and electrophoretic gel shift assays. The results suggest that CP2 binds a hyphenated recognition sequence motif that spans one DNA helix turn. In addition, the enriched bacterial protein activated transcription of alpha-globin promoter templates approximately 3- to 4-fold in vitro. We then tested the effect of elevating CP2 levels 2.5- to 5.5-fold in vivo using both transient and stable transformation assays. When a reporter construct comprised of the intact murine alpha-globin promoter driving the bacterial chloramphenicol acetyltransferase (CAT) gene was introduced into these overexpressing cells, we observed a 3- to 6-fold increase in CAT activity when compared to cells expressing normal levels of CP2. These results define the CP2 factor binding site in more detail and help characterize the activities of the factor in vivo.