The distribution, structural organization and state of phosphorylation of neurofilaments have been examined in chromaffin cells from adult bovine adrenal medulla cultured under various conditions using a series of monoclonal antibodies directed against phosphorylated and nonphosphorylated epitopes of the 200,000 mol. wt subunit. Nonphosphorylated neurofilament epitopes were detected immunocytochemically to varying extents in chromaffin cells maintained under standard culture conditions for up to 3 weeks. Staining was usually limited to a perinuclear region from which fine filaments sometimes appeared to radiate around the nucleus. In marked contrast, none of the antibodies directed against phosphorylated neurofilament epitopes stained these structures. When cells were cultured under conditions favouring neurite outgrowth, in conditioned medium derived from intermediate lobe cultures, there was a more extensive expression of the nonphosphorylated neurofilament epitopes. In addition, phosphorylation of neurofilaments was induced. The phosphorylated neurofilament epitopes were restricted to the neurite, whereas the nonphosphorylated neurofilament epitopes were localized in both neurite extensions and perikarya. These results demonstrate that conditioned medium from intermediate lobe cells of the hypophysis not only provokes neurite outgrowth from chromaffin cells, but also supports neuronal maturation as demonstrated by the phosphorylation of neurofilaments in neurites.