**Abstract**

Cyclic adenosine monophosphate (cAMP), which is derived from adenosine triphosphate through adenylyl cyclase A (acaA), acts as an intracellular secondary messenger and an extracellular chemotactic substance in important biological processes. In the social amoebae *Dictyostelium discoideum*, cAMP mediates cell aggregation, development, and differentiation to spore and stalk cells during fruiting body formation. The *acaA* gene is transcribed under the control of three different alternative promoters. This study aimed to develop a promoter assay for *acaA* in *D. discoideum* using bioluminescence microscopy. Here, we inserted green- and red-emitting luciferase genes into downstream of promoter regions 1 and 3, respectively. Promoter activities were visualized by bioluminescence microscopy. We confirmed the differential expression of *acaA* under the control of promoters 1 and 3 at the different stages of *D. discoideum* development. We also demonstrated the application of dualcolor bioluminescence imaging in the development of an imaging promoter assay.

**Keywords:** *Dictyostelium discoideum*, adenylyl cyclase A promoter, dual-color luciferase, bioluminescence microscopy, imaging promoter assay, fruiting body formation
