A number of the benzodiazepines (BZDs) inhibit nerve growth factor (NGF)-induced neurite outgrowth in a dose-dependent manner in PC12 cell cultures. The rank order of potency of a series of BZDs for inhibition of neurite outgrowth does not correlate with the order of their affinity constants for the so-called peripheral BZD sites present on PC12 cells. Whereas the inhibition of neurite extension is stereospecific, the binding to the peripheral site is not. The inhibition of neurite outgrowth is not attributable to a blockade of NGF binding to either its fast or slow receptors. Additionally, several other characteristics of NGF stimulation of PC12 cells, such as autoadhesion and the induction of ornithine decarboxylase (OrnDCase), remain unaltered in the presence of BZDs. Many BZDs increase OrnDCase levels in PC12 cells in the absence of NGF. The OrnDCase response to BZDs is blocked by actinomycin D. Furthermore, the structural requirements of BZDs for induction of OrnDCase activity is not identical to that for the inhibition of NGF-induced neurite extension. Thus, unlike attenuation of neurite extension, BZD induction of OrnDCase is not stereospecific. Also, some BZDs block neurite growth but do not induce OrnDCase. We propose that there are at least three sites of action of BZDs on PC12 cells. The first is the well-characterized high-affinity peripheral site. The second distinct locus of action results in a blockade of NGF-induced neurite outgrowth. The structural requirements for the latter effect are essentially indistinguishable from those for BZD induction of hemoglobin synthesis in Friend erythroleukemia cells. The third site of action results in an induction of OrnDCase. Structural requirements for this activity are not identical to those for either the differentiation of Friend cells or the inhibition of neurite outgrowth.