Neurotrophins are required for the differentiation and survival of several different neuronal subpopulations in the developing nervous system. The PC12 cell line responds to nerve growth factor (NGF) by withdrawing from the cell cycle and acquiring a sympathetic neuron-like phenotype. Previous studies have shown that the activation kinetics of the NGF receptor, TrkA, and downstream protein kinases appear rapid and seemingly transient after NGF treatment of naive PC12 cells. However, maintenance of the neuronal phenotype and survival of differentiated PC12 cells under serum-free conditions require constant NGF exposure. In this study we have addressed the mechanisms that NGF uses to maintain neuronal PC12 cells. We show that TrkA remains phosphorylated at a basal level throughout differentiation of the PC12 cells. The phospho-TrkA levels in the differentiated PC12 cells were diminished by both complete NGF withdrawal and pharmacological inhibition of Trk kinase activity. Intracellular sequestration of the majority of TrkA molecules (both phosphorylated and non-phosphorylated TrkA) and persistent dephosphorylation of the small pool of cell surface TrkA renders the persistent phospho-TrkA signal in the differentiated PC12 cells resistant to partial NGF withdrawal as well as exposure to additional NGF. NGF regulated both extracellular-regulated kinases 1/2 and Akt activity in the differentiated PC12 cells via sustained TrkA activity. Moreover, analysis of transcription using activating protein 1-, serum response element-, and cyclic AMP response element-Luc reporter constructs showed that NGF regulated these promoters through TrkA activity in differentiated PC12 cells. Interestingly, the initial response of the cyclic AMP response element promoter to NGF was delayed, becoming Trk-dependent well beyond the peaks in TrkA and downstream protein kinase signal transduction.