Continuously cultured human neuroblastoma cell lines SK-N-SH, SK-N-BE(1), SK-N-BE(2), and SK-N-MC, as well as several clonally derived sublines, were assessed for their neurotransmitter characteristics. Two different methods were used: measurement of cholinergic and adrenergic enzyme activities and detection of neurotransmitters synthesized from radioactive precursors. Dopamine-β-hydroxylase (EC 1.14.2.1), choline acetyltransferase (EC 2.3.1.6), acetylcholinesterase (EC 3.1.1.7), and butyrylcholinesterase (EC 3.1.1.8) levels were compared to those of various normal and neuronal cell controls. The capacity of neuroblastoma lines and clones to convert radioactive tyrosine, choline, and glutamate to transmitter products was determined. SK-N-SH, SK-N-BE(1), and SK-N-BE(2) cells had moderately high levels of dopamine-β-hydroxylase activity. Whereas neuroblast-like clones of SK-N-SH had similar levels of this enzyme, epithelial-like clones had no detectable activity. SK-N-BE(2) cells showed the greatest capacity to convert tyrosine to dopamine. Although the SK-N-SH line itself was not tested, both neuroblast and epithelial-like clones, shown by karyotype analysis to have a common cell precursor, converted tyrosine to dopamine. In contrast, the SK-N-MC line and its clones appeared devoid of adrenergic properties. All cells tested except human fibroblast-like cells of normal origin were able to convert radioactive choline to acetylcholine. While all the cell lines had measurable choline acetyltransferase activity, levels were substantially higher (at least 4- to 12-fold) for SK-N-MC cells and its clonally derived subline MC-IXC. All cells tested converted glutamate to γ-aminobutyric acid. Results indicate that these human neuroblastoma cell lines have predominating neurotransmitter characteristics that are sometimes, but not always, expressed in clones derived from them. Since the SK-N-BE(1) and SK-N-BE(2) lines were isolated 5 months apart from the same patient and have similar activity levels of dopamine-β-hydroxylase, these characteristics may be stable and representative of the tumor cells in vivo. Furthermore, results obtained by the two different methodological approaches suggest that, whereas SK-N-MC cells show cholinergic and not adrenergic traits, SK-N-SH and possibly SK-N-BE(2) may be multipotential with regard to neuronal enzyme expression.