The promoter of α subunit of the rat calcium/calmodulin-dependent protein kinase II (αCaMKII) gene was identified to contain an essential TATA element. Cell-based functional assay showed that the rat promoter displayed greater activity in neuronal cells than in non-neuronal cells. To characterize the human αCaMKII promoter, we have developed a promoter-reporter gene assay using different cell lines. A 2047 base pairs (bp) human αCaMKII gene promoter was cloned from human genomic DNA. Unlike the rat αCaMKII promoter, DNA sequence analysis showed that the human promoter was devoid of TATA element. We made series deletions of the promoter and fused the different sizes of the human promoter sequences to a luciferase reporter gene. The promoter-reporter constructs were transfected into human neuroblastoma SH-SY5Y, human neuroblastoma BE(2)-M17, and rat pheochromocytoma PC12 neuronal cell lines as well as human embryonic kidney HEK293 and human glioma U251 non-neuronal cell lines. The reporter gene assay demonstrated that the human αCaMKII promoter displayed high activity in the neuronal cell lines, while the activity was low in non-neuronal cell lines. All-trans retinoic acid (RA) enhanced the promoter activity in SH-SY5Y cells. Further analysis showed that there were two RA response elements located between +11 and +136 and −1911 to −593. In addition, we have identified a potent silencer at position −179 to −244 of the human αCaMKII promoter.