The use of bacterial artificial chromosome (BAC) reporter constructs in molecular physiology enables the inclusion of large sections of flanking DNA, likely to contain regulatory elements and enhancers regions that contribute to the transcriptional output of a gene. Using BAC recombineering we have manipulated a 160kb human prolactin luciferase (hPRL-Luc) BAC construct and mutated the previously defined proximal estrogen response element (ERE) located -1189bp relative to the transcription start site, to assess its involvement in the estrogen responsiveness of the entire hPRL locus. We found that GH3 cell lines stably expressing luciferase under control of the ERE-mutated hPRL promoter (ERE-Mut) displayed a dramatically reduced transcriptional response to E2 treatment compared to cells expressing luciferase from the wild type (WT) ERE hPRL-Luc promoter (ERE- WT). The -1189 ERE controls not only the response to E2 treatment, but also the acute transcriptional response to TNFalpha, which was abolished in ERE-Mut cells. ERE-WT cells displayed a biphasic transcriptional response following TNFalpha treatment, the acute phase of which was blocked following treatment with the estrogen receptor antagonist 4-hydroxytamoxifen (4-OHT). Unexpectedly we show the oscillatory characteristics of hPRL promoter activity in individual living cells were unaffected by disruption of this crucial response element, as real-time bioluminescence imaging showed transcription cycles were maintained, with similar cycle lengths, in ERE-WT and ERE-Mut cells. These data suggest the -1189 ERE is the dominant response element involved in the hPRL transcriptional response to both E2 and TNFalpha and crucially, that cycles of hPRL promoter activity are independent of ER binding.