Neuroendocrine (NE) differentiation within prostate tumors is proposed to be a contributing factor in disease progression. However, the cellular origin and molecular mechanism controlling differentiation of prostatic NE cells are unresolved. The prostate tumor cell line, LNCaP, can reversibly acquire many NE characteristics in response to treatment with beta-adrenergic receptor agonists and activators of adenylate cyclase. In this study, we demonstrate that these treatments induce protein kinase A (PKA) activation in LNCaP cells and that ectopic expression of a constitutively activated form of the PKA catalytic subunit, CIalpha, results in acquisition of NE characteristics, including the extension of neuritic processes, cessation of mitotic activity, and production of neuron-specific enolase. Forskolin-, epinephrine-, and isoproterenol-dependent NE differentiation of LNCaP cells was significantly inhibited by expressing a dominant negative mutant of the PKA regulatory subunit, RIalpha. These results demonstrate that prostatic NE differentiation in response to these agents depends on PKA activation, and this signaling pathway may provide a therapeutic target for treating advanced forms of prostate cancer.