The promoters of three juvenile hormone (JH)-sensitive, and one JH-insensitive hexamerin-encoding genes (Hex) were isolated from Trichoplusia ni, and sequences necessary for, or affecting, transcriptional activity were identified by biochemical and functional methods. The transcription start points (tsp) for each of the four Hex were determined biochemically, by both primer extension and the sequencing of multiple, independent full-length cDNA clones. The function of each inferred tsp, as an actual tsp, was confirmed by in vitro transcription assay. The transcription initiator sequence, GNACAGT, was identical for three of the Hex, while the fourth used a divergent motif. Using the in vitro transcription system, a minimal core promoter of 60 bp (bp -34 to +24) of the BJHSP1 (basic JH suppressible protein 1) gene, containing a single TATA box motif approximately 30 bp upstream of the tsp, was functionally sufficient to support alpha-amanitin-sensitive transcription. The same construct was also transcriptionally functional in a homologous cell line transfection assay. The corresponding region of the other Hex also contains a similarly positioned TATA box motif, and promoter constructs for each, that included that included the tsp, initiator and inferred basal transcription apparatus binding site, were all transcriptionally functional in a cell line transfection assay. The action of sequences 5' to the minimal promoter region in modulating the rate of transcription was shown by a cell line transfection assay of a nested deletion series of the promoter for the BJHSP1 gene, in which the results identified a strongly suppressive element between positions -160 and -109. This system of Hex genes, including those sensitive to JH and one not sensitive, should be useful in a comparative approach toward identifying those regulatory motifs that are functionally necessary to transduce the regulatory action of JH.