Morphological characteristics of undifferentiated and differentiated human neuroblastoma cells were studied. Monolayer cultures of a human neuroblastoma, IMR-32 clone, were grown in Eagle's minimum essential medium with fetal calf serum in tissue culture dishes with polystyrene film liners. After 48 h, cultures were treated with either mitomycin C and 5-bromodeoxyuridine or prostaglandin E1 (PGE1) and dibutyryl adenosine 3',5'-cyclophosphate (cAMP). A third dish was untreated to study as an undifferentiated control. Three days later, all cultures were processed for acetylcholinesterase staining, scanning and transmission electron microscopy and high performance liquid chromatography. Treatment with mitomycin/5-bromodeoxyuridine and PGE1/cAMP inhibited growth as seen by the growth curves and caused morphological differentiation as seen by the extension of long neurites. The treated cells showed increased acetylcholinesterase staining compared to the controls. With the scanning electron microscope, the differentiated cells showed long neurites, processes with beaded varicosities and growth cones. By transmission electron microscopy, these cells contained a large number of neurosecretory granules in their cytoplasm and neurites. Specialized cell contacts were also observed between the treated cells. This is the first study demonstrating that both the treated and control cells of IMR-32 clone contain large quantities of serotonin and comparatively small amounts of norepinephrine and dopamine.