Correct developmental regulation of beta-like globin gene expression is achieved by preferential transcription of a gene at a given developmental stage, silencing of other beta-like gene promoters, and competition among these promoters for interaction with the locus control region (LCR). Several evolutionarily conserved DNA elements in the promoters of the beta-like genes and LCR have been studied in detail, and the role of their binding factors has been investigated. However, the beta-globin promoter includes additional evolutionarily conserved sequences of unknown function. The present study examined the properties of a 21-base pair (bp) promoter-conserved sequence (PCS) located at positions -115 to -136 bp relative to the transcription start site of the beta-globin gene. A helicaselike transcription factor (HLTF) belonging to the SWI2/SNF2 family of proteins binds to the PCS and a partly homologous sequence in the enhancer region of the LCR hypersensitive site 2 (HS2). Elevation of the level of HLTF in K562 erythroleukemic cells increases beta-promoter activity in transient transfection experiments, and mutations in the PCS that remove HLTF-binding regions abolish this effect, suggesting that HLTF is an activator of beta-globin transcription. Overexpression of HLTF in K562 cells does not affect the endogenous levels of gamma- and epsilon-globin message, but it markedly activates beta-globin transcription. In conclusion, this study reports a transcription factor belonging to the SWI2/SNF2 family, which preferentially activates chromosomal beta-globin gene transcription and which has not previously been implicated in globin gene regulation.