In the milkweed bug, Oncopeltus fasciatus, the classic ecdysone early response transcription factor E75 also plays an essential role in segmental patterning during early embryogenesis. Using degenerate primers targeting the conserved DNA binding domain (DBD) of the nuclear hormone receptor family, E75 was found to be expressed within the first 24 hr of embryogenesis. Two isoforms, E75A and E75B, were subsequently obtained via RACE amplification of day 2 embryonic cDNA. In situ hybridization showed early E75 mRNA expression in transverse stripes on the blastoderm likely corresponding to gnathal and thoracic segments: two at 24 hours and four just prior to invagination at 30 hrs. During germ band formation in this intermediate-germ insect, E75 mRNA appears in the growth zone during abdominal elongation. Injections of both the E75-DBD and E75A-specific RNAi into females produced embryos that lacked the second thoracic (T2) and labial (Lb) segments and, in more affected animals, was accompanied by the loss of T3 legs and several abdominal segments. Hox gene analysis in the germ band stage demonstrate that E75 is important for specifying parasegments in the Oncopeltus blastoderm. For example, proboscipedia mRNA, which is normally restricted to the Lb appendages in the germ band, appears in the anterior half of the apparently fused Lb and T1 appendages. Epistatic relationships to other early patterning genes are being studied. Supported by NIH GM 060122. HK and DFE contributed equally to the work.