Homologous transfection systems provide a useful tool for characterizing promoters and other regulatory elements from cloned genes. We have used cultured Aedes albopictus C7–10 mosquito cells to evaluate expression of 20-hydroxyecdysone-inducible genes. Although this cell line has previously been shown to synthesize components of the ecdysteroid receptor and ecdysone-inducible proteins, the well-characterized ecdysteroid response element (EcRE) from the Drosophila hsp27 promoter failed to confer a substantial 20-hydroxyecdysone mediated induction in transfected mosquito cells. Recovery of stably transformed clones was also reduced in a DNA dependent manner when the EcREs were in the sense orientation, relative to control plasmids lacking the EcREs or containing an antisense construct. Finally, when tandem EcREs were placed within the hsp70 promoter, CAT activity was detected only after prolonged enzyme incubation, suggesting that the DNA interfered with cellular metabolism. In these constructs, we noted that the promoter DNA contained several potential binding sites for the activator protein-1 (AP-1) transcription factor, one of which lay between the tandem EcREs. On southwestern blots, a 40 kDa nuclear protein from C7–10 cells bound to DNA containing AP-1 sites. A DNA affinity column was used to partially purify the 40 kDa protein, and western analysis showed that the mosquito protein cross-reacted with a heterologous antibody to JUN. Likewise, mRNA from C7–10 cells cross-hybridized with the jun cDNA from Drosophila. These results suggest that like estrogen, 20-hydroxyecdysone interfaces with AP-1 as a co-activator protein that modulates the overall hormone response.