When grown for seven days in a medium containing nerve growth factor (100 ng/ml), 10% horse serum and 5% fetal bovine serum PC12 cells stopped dividing, extended neurites and assumed a neuronal phenotype. Withdrawal of nerve growth factor from these cells resulted in loss of neurites and apoptotic changes in many cells. The apoptotic changes were exacerbated if the cells were also exposed to 1-2 microM S-100, a calcium binding protein purified from bovine brain. After exposure to S-100, the PC12 cells underwent characteristic apoptotic changes. Within 2 in neurites retracted, the cell body shrunk and submembranous accumulation of condensed cytoplasmic material was observed. DNA ladders were present after 24-48 h and 60% of the cells became hypodiploid after 72 h. S-100 induced apoptosis by binding to specific sites (Kd = 189 nM) on PC12 cells and this caused a rise in [Ca2+]i due to a transmembrane capacitative flux followed by the depletion of internal stores. This increase was reversed if 5 microM nifedipine, a specific L-type Ca2+ channel inhibitor, was added to the medium after S-100 and completely abolished if the cells were pretreated with 5 microM thapsigargin, an inhibitor of endoplasmic reticulum Ca(2+)-ATPase. The presence of nerve growth factor in the culture medium completely blocked the apoptotic changes induced by S-100, probably due to interaction of nerve growth factor and S-100 at the same binding sites. These data indicate that nerve growth factor not only prevents apoptosis during cell development, but also apoptosis induced by endogenous substances such as S-100.