NSC-34 cells differentiated into motor neurons (MN) by exposure to retinoic acid (RA) are widely used as an experimental model of MN. We reported previously that prostaglandin E₂ (PGE₂) also differentiates these cells and promotes neurite outgrowth, a morphological marker of neuronal differentiation. However, it still remains unclear whether PGE₂-treated cells possess characteristics of mature MN. In this study, we compared the biochemical and functional properties of PGE₂-treated NSC-34 cells with those of RA-treated cells. PGE₂ (30 μM) induced neurite outgrowth which reached a peak at day 2, whereas RA (10 μM) -induced neurite outgrowth reached the same level at day 7. Immunoblotting showed that expression levels of a neuronal marker (Synaptophysin) and MN markers (HB9 and Islet-1) in PGE₂-treated cells at day 2 were comparable to those in RA-treated cells at day 7. The level of choline acetyltransferase protein and the basal acetylcholine release in PGE₂-treated cells were higher than those in RA-treated cells. These results suggest that both PGE₂ and RA promote neuronal maturation of NSC-34 cells, but PGE₂ is more effective to promote cholinergic function in these cells than RA.