Ectopic expression of the homeobox gene HOX11 is associated with a significant proportion of childhood T-cell acute lymphoblastic leukaemias (T-ALLs). We hypothesise that one mechanism of gene deregulation involves overcoming the silencing mechanism(s) of gene expression present in normal cells. Here, we describe a search for trans-acting factors that control transcriptional activity from a distal 5' region of the HOX11 promoter. We have identified a region of this promoter which contributes significantly to HOX11 activation and two distinct regulatory elements are involved. First, a PBX2 Regulatory Element PRE-1048 has been identified which contains a novel DNA-binding sequence and mediates significant activation of the HOX11 gene in K562 cells. This is the first report of a homeobox gene being specifically regulated by PBX2 and the second report of a vertebrate homeobox target gene of a PBX protein. The PREP1 protein was also shown to be part of the PRE-1048-binding complex. The other regulatory element we describe here RE-1019 contains little sequence conservation to known transcription control elements. It appears that this element is a novel sequence that binds an as yet unidentified factor, mediating significant activation of the HOX11 gene in K562 cells. This is the first detailed report of elements that mediate regulation of the proto-oncogene HOX11.