Objective:To construct luciferase reporter plasmids of full-length and truncated promoters of growth arrest- and DNA-damage-inducible gene 45 (Gadd45) β/γ and detect their activity in rat GMCs in response to activating transcription factor 3 (ATF3) overexpression respectively, screening the possible binding sites for ATF3. Methods: Rat Gadd45β promoter (-110 5 ~ +236 nt) and Gadd45γ promoter (-913 ~ +72 nt) were amplified by PCR, and then cloned into the luciferase reporter plasmids (pGL3-basic) separately. The recombinant plasmids (pGL3-Gadd45β/γ-FL) were transfected into GMCs accompanied with rat ATF3 expression plasmid (pIRES2/ATF3) and the luciferase activity was detected to determine the role of ATF3 in Gadd45β/γ gene transcription. Meanwhile, the potential ATF3 binding sites within Gadd45β/γ promoter were predicted by bioinformatics software. Based on the predicted results, four truncated Gadd45β promoter luciferase reporters and three truncated Gadd45γ promoter luciferase reporters were constructed. Subsequently, the full-length as well as different truncated promoter luciferase reporters of Gadd45β/γ were transfected into GMCs accompanied with pIRES2/ATF3, and the luciferase activity was then detected to identify the ATF3 binding sites. Results: It was verified that different kinds of plasmids were all constructed correctly by PCR analysis and nucleotide sequencing. GMCs co-transfected with pGL3-Gadd45β/γ-FL and pIRES2/ATF3 displayed increased luciferase activity of Gadd45β and Gadd45γ promoter, respectively. In addition, pGL3-Gadd45β-4 co-transfected with pIRES2/ATF3 in GMCs showed notably reduced Gadd45β promoter activity than that of pGL3-Gadd45γ-FL, pGL3-Gadd45β-1, pGL3-Gadd45β-2, pGL3-Gadd45β-3 co-transfected with pIRES2/ATF3, indicating that the region of rat Gadd45β promoter (-146 ~ +23 nt) might contain ATF3 binding element. Likewise, pGL3-Gadd45γ-2 and pGL3-Gadd45γ-3 co-transfected with pIRES2/ATF3 in GMCs exhibited acute reduction of Gadd45γ promoter activity than that of pGL3-Gadd45γ-FL and pGL3-Gadd45γ-1, indicating that the region of rat Gadd45γ promoter (-456 ~ -61 nt) might contain more than one ATF3 binding element. Conclusion: The full-length and truncated Gadd45β/γ promoter luciferase reporter plasmids were constructed successfully, and the ATF3 binding regions within Gadd45β/γ promoters were identified.