1,25-Dihydroxyvitamin D₃ (1,25(OH)₂D₃), the active metabolite of vitamin D, induces nerve growth factor (NGF) synthesis in a variety of different cell lines. The mechanism by which 1,25(OH)₂D₃ induces NGF, however, is poorly understood. We used a series of full-length and truncated NGF promoter−human growth hormone (hGH) reporter gene plasmids to investigate the mechanism of 1,25(OH)₂D₃-induced NGF expression in osteoblasts. Untransfected rat osteosarcoma cells (ROS 17/2.8) treated with 1,25(OH)₂D₃ showed a 2-fold increase in NGF expression compared to control cells. ROS 17/2.8 osteosarcoma cells were transfected with the NGF−hGH reporter plasmids and treated with 10^-8 M 1,25(OH)₂D₃. The full-length NGF promoter (−1800 to +120)−hGH reporter construct showed an approximately 2-fold increase in hGH release. Plasmids with successive 5‘-deletions showed enhanced hGH expression in treated cells and control cells. A similar series of NGF promoter−hGH reporter gene constructs, lacking the AP-1 site located within the first intron of the NGF gene, were also transiently transfected into ROS 17/2.8 cells. When these cells were treated with the same dose of 1,25(OH)₂D₃, no increase in hGH expression was seen compared to control cells, demonstrating that this AP-1 site is essential for 1,25(OH)₂D₃-mediated NGF up-regulation. Since 1,25(OH)₂D₃ is known to activate the transcription of several genes through its interaction with the vitamin D receptor (VDR), we performed a series of gel electrophoretic mobility shift assays to determine if the VDR binds directly to the AP-1 sequence. No evidence of VDR binding, either as a homodimer or as a heterodimer, to the AP-1 sequence was observed. Treatment of ROS 17/2.8 cells with 1,25(OH)₂D₃, however, resulted in an increase in AP-1 binding activity; however, no significant changes in c-jun and c-fos levels were observed. Our data show that in osteoblasts, 1,25(OH)₂D₃ induces NGF expression indirectly by increasing AP-1 binding activity.