This suggests that the mechanism engaged to repair a DSB is a function of the cell cycle stage at which repair is executed, rather than the stage at which the DSB itself is induced. The rare-cutting restriction endonuclease, I-SceI, has become a major tool for analyzing mechanisms of DSB repair. I-SceIinduced breaks are likely limited to the canonical I-SceI target site and the DSB ends generated by I-SceI-mediated breakage are defined by the enzyme’s known endonuclease activity. In contrast, IR-induced DSBs are distributed across the genome and the DSB ends may be chemically modified. For these reasons, the “rules” governing I-SceI-induced DSB repair might differ from those governing an IR-induced DSB. The ligand-binding domain of the estrogen receptor has been used to regulate the activity of a variety of nuclear proteins, since fusion proteins containing the ER LBD are retained in the cytoplasm until activated by 17ß-estradiol. Ligand binding facilitates correct folding of the released fusion protein, thereby activating the LBD-fused nuclear protein. The development and characterization of a system to control ISceI expression in a tight temporal fashion is an important research tool for the study of DSB repair. Its applications may include the study of cell cycle relationships, as discussed above, to potential use in chromatin immunoprecipitation studies, realtime imaging, high-throughput screening and tight control of ISceI activity in animal models. The accurate interpretation of siRNA experiments might also benefit from a tractable inducible DSB system such as that described here, where DSB induction could be initiated when siRNA knockdown is at its peak. The use of a recombination reporter is a stringent test of the activity of the enzyme – probably more so than attempts to directly measure breakage at the locus. Notably, cultivation of cells in the presence of SP600125 phenol red and endogenous estrogens in serum that was not charcoal stripped, triggered the gradual accumulation of GFP + products of I-SceI-induced HR in the absence of 4OHT. This suggests that, if residual estrogens are present in the culture medium, they can produce some basal activation of the fusion protein, even in its optimal “EIE” configuration. This suggests that the application of this technology in animal models may require additional levels of control. In this report, we have identified an optimal configuration of ER-LBD-I-SceI fusion protein that provides tight control of I-SceI activity in the absence of activating ligand. Our experience matches closely that described for ER LBD control of the Cre recombinase and the results of Bennardo et al. in control of ISceI. In each, the addition of ER LBD domains to both the N- and C-termini of the enzyme provide optimal control with retention of enzymatic activity. This result is also consistent with the crystal structure of I-SceI.