Tandem insertions of defective P elements (1.15 kb KP and 0.6 kb core P) accelerate the transcription rate of the glucose-6-phosphate dehydrogenase (G6PD) gene in Drosophila melanogaster. In this report, we have analyzed the activation mechanism of the G6PD promoter by in vitro transcription and gel retardation assays. Results showed that one cis-acting region in the core P and two such regions in the KP are associated with activation of the G6PD promoter, and that putative transcriptional regulatory protein(s) which specifically bind to each of the cis-acting regions are present in nuclear extracts of Canton S embryos. On the other hand, the P elements do not activate the normal actin 5C promoter, but activate the promoter when the 20 bp sequence around the G6PD transcription start site is placed in front of the promoter. It appears that the GC-rich region in this 20 bp sequence is required for the activation.