For Drosophila melanogaster flies, sexual fate is determined by the X chromosome number. The basic helix-loop-helix protein product of the X-linked sisterlessB (sisB or scute) gene is a key indicator of the X dose and functions to activate the switch gene Sex-lethal (Sxl) in female (XX), but not in male (XY), embryos. Zygotically expressed sisB and maternal daughterless (da) proteins are known to form heterodimers that bind E-box sites and activate transcription. We examined SISB-Da binding at Sxl by using footprinting and gel mobility shift assays and found that SISB-Da binds numerous clustered sites in the establishment promoter SxlPe. Surprisingly, most SISB-Da sites at SxlPe differ from the canonical CANNTG E-box motif. These noncanonical sites have 6-bp CA(G/C)CCG and 7-bp CA(G/C)CTTG cores and exhibit a range of binding affinities. We show that the noncanonical sites can mediate SISB-Da-activated transcription in cell culture. P-element transformation experiments show that these noncanonical sites are essential for SxlPe activity in embryos. Together with previous deletion analysis, the data suggest that the number, affinity, and position of SISB-Da sites may all be important for the operation of the SxlPe switch. Comparisons with other dose-sensitive promoters suggest that threshold responses to diverse biological signals have common molecular mechanisms, with important variations tailored to suit particular functional requirements.