The cry1Ac gene of Bacillus thuringiensis subsp. kurstaki HD-73 (B. thuringiensis HD-73) is a typical example of a sporulation-dependent crystal gene and is controlled by sigma E and sigma K during sporulation. To monitor the production and accumulation of Cry1Ac at the cellular level, we developed a green fluorescent protein-based reporter system. The production of Cry1Ac was monitored in spo0A, sigE, and sigK mutants, and these mutants were able to express the Cry1Ac-green fluorescent protein fusion protein. In nonsporulating B. thuringiensis HD-73 cells, low-level expression of cry1Ac was also observed. Reverse transcription-PCR and Western blotting results confirmed that the cry1Ac promoter has low activity in nonsporulating B. thuringiensis cells. A beta-galactosidase assay demonstrated that the transcription of the cry1Ac gene during exponential and transition phases is positively regulated by Spo0A. Additional bioassay results indicated that spo0A and sigE mutants containing the cry1Ac-gfp fusion exhibited insecticidal activity against Plutella xylostella larvae.