Our previous studies on the human beta globin gene cluster revealed the presence of intergenic transcripts throughout the locus, and demonstrated that transcription of the locus control region (LCR) initiates within an ERV9 endogenous retroviral long-terminal repeat (LTR) upstream of DNase I hypersensitive site 5. We show, using a combination of assays, that there are additional sites of transcription initiation within the LCR at hypersensitive sites 2 and 3. We have defined sites of transcription initiation, which occurs at discrete positions in a direction towards the globin genes. In addition, we show that mutation of specific transcription factor binding sites within HS2 leads to a reduction in transcription levels from within this site. We propose that these initiation events within the LCR can account for the observed orientation dependence of LCR function, and contribute to the open chromatin configuration of the beta globin locus. In addition, transcription from within the LCR hypersensitive sites could compensate for the absence of the ERV9 LTR in many transgenic mice lines, which nevertheless regulate their globin clusters correctly.