Comparative protein binding studies have been performed on the Xenopus beta globin gene promoter. Erythroblast nuclear extracts 'footprint' over the erythroid-specific consensus sequence, AGGATAAG, which is located immediately upstream of the CCAAT footprint. Nonerythroid cell extracts do not give rise to an AGGATAAG footprint but rather to an extended CCAAT footprint reminiscent of the CCAAT displacement protein (CDP). Erythroblast extracts also protect a sequence similar to the chicken stage selector element (SSE) immediately downstream of the CCAAT box footprint. In contrast to these discrete footprints observed using erythroblast extracts, Xenopus erythrocyte nuclear extracts give rise to more extensive promoter protection. We have previously reported that this promoter is active in transfected HeLa cells when linked to the SV40 enhancer and that transcriptional activation is accompanied by the formation in the chromatin of a nuclease hypersensitive site (HS) in this region. As a first step towards defining the roles of the various promoter-binding proteins in transcriptional activation and HS formation, we transfected deletion mutants of the promoter into HeLa cells. Deletion of the sequences upstream of -116 had no effect on transcription or HS formation. Indeed the upstream boundary of the HS remained unchanged (at around-170) even though plasmid sequences had replaced Xenopus sequences. If the HS boundary reflects resumption of nucleosomal structure, then sequences downstream of -116 must be able to position a nucleosome from at least 50 bp away. beta globin gene activation in a number of transfected cell lines is absolutely dependent on DNA replication. The replication requirement is not a consequence of template copy number or methylation, nor is it dependent on the direction in which the replication fork passes through the gene. We conclude that replication facilitates active transcription complex formation by disrupting a stable association of the template with negative factors, which could include histones. About 200 bp upstream of the Xenopus beta globin gene promoter is a tract of alternating A and T residues which adopts cruciform geometry at low levels of supercoiling. Because of this sensitivity to torsional stress, we have probed the structure of the (AT)n sequence in microinjected Xenopus oocytes, where the Xenopus beta globin gene is transcribed very efficiently. We find that S1 nuclease cleaves specifically in the middle of the (AT)n tract, suggesting that the gene is under torsional stress.