Although DNA transfection with calcium-phosphate/DNA precipitates for promoter analysis of genes has been previously applied to primary cultures of neuronal cells, it remains uncertain whether the expression of the introduced genes in glial cells, which are also in primary culture, affects the transcriptional signals obtained. Using plasmid DNA containing the brain-derived neurotrophic factor (BDNF) gene promoter I or c-fos promoter, we investigated the optimum conditions for calcium-phosphate-mediated DNA transfection in primary culture of rat cortical neuronal cells. Normalizing the firefly luciferase activity of the experimental reporter gene to the Renilla luciferase activity of the internal control increased experimental reliability. Maximum expression of firefly luciferase activity in the cells stimulated by membrane depolarization required 6-12 hrs of incubation after stimulation. Differences in the ratio of the experimental reporter gene to the internal control did not affect experimental gene expression. Under our optimal conditions, the activation of the BDNF gene promoter I was detected in neuronal but not in glial cells. Calcium-phosphate-mediated DNA transfection should be widely applicable for promoter analysis of inducible genes in neurons.