The immediate-early gene NGFI-B (also called nur77) encodes an orphan nuclear receptor that activates transcription through a unique response element (NBRE). NGFI-B is rapidly induced and modified via phosphorylation by a variety of stimuli that induce cells to differentiate or to proliferate. We have shown that the in vitro phosphorylation of Set350 located within the 'A-box,' a motif necessary for DNA binding by NGFI-B, results in a decrease in the binding of NGFI-B to its response element (Hirata, Y., Kiuchi, K., Chen, H.-C., Milbrandt, J., and Guroff, G. (1993) J. Biol. Chem. 268, 24808- 24812). We show here that nerve growth factor (NGF)-induced changes in the in vivo phosphorylation of Set350 accompany transcriptional deactivation of NGFI-B in PC12 cells, that membrane depolarization and NGF treatment cause differential phosphorylation of NGFI-B, and that the transcriptional activation caused by exogenous expression of NGFI-B or membrane depolarization can be inhibited by NGF treatment. In addition, the mutation of Set350 to Ala abolished the inhibitory effect of NGF on the transcriptional activation of NGFI-B in PC12 cells. These data could provide new insights into the regulation of transcriptional activity required for some neurons to switch from activity-dependent survival to neurotrophin- dependent survival during development.