Juvenile hormone binding protein (JHBP) is the key element of the system that transmits hormone signals to target tissues. Recently, we found that the core promoter of the jhbp gene is strongly under the control of the TATA box and the transcription start site. In this report, we have shown that the jhbp promoter contains distal regulatory elements whose functionality clearly depends on the particular cell environment and that the scope of research from one cell line is insufficient to generalize the conclusions of the analysis. Cf1/Usp (where Usp is ultraspiracle protein previously known as Cf1, chorion factor 1) elements suppressed transcription of the reporter gene in the High Five cell line but not in the Sf9 cell line. However, upstream from all three Cf1/Usp elements there is a DNA sequence, containing the Zeste element, which activates jhbp in both systems. We found that juvenile hormone strongly inhibited the activity of the jhbp promoter in the Sf9 cell line, whereas it did not have an effect in the High Five cell line. A second key hormone that controls insect development--20-hydroxyecdysone, was also found to suppress the transcription of jhbp. This is the first report describing how these two hormones affect jhbp gene expression in different cell lines.