, BDNF-PI was markedly activated, as well as BDNF-PIII, by Ca 2؉ signals evoked via neuronal activity. However, little is known about the mechanisms for the transcriptional activation of BDNF-PI. Using rat cortical neurons in culture, we assigned the promoter sequences responsible for the Ca 2؉ signal-mediated activation of BDNF-PI and found that the Ca 2؉ -responsive elements were located in two separate (distal and proximal) regions and that the DNA sequences in the proximal region containing cAMP-responsive element (CRE), which is overlapped by the upstream stimulatory factor (USF)-binding element, were largely responsible for the activation of BDNF-PI. CRE-binding protein (CREB) family transcription factors and USF1/USF2 bind to this overlapping site, depending upon their preferred sequences which also control the magnitude of the activation. Overexpression of dominant negative CREB or USF reduced the BDNF-PI activation. These findings support that not only CREB but also USF1/USF2 contributes to Ca 2؉ signal-mediated activation of BDNF-PI through the recognition of an overlapping CRE and USF-binding element.