Some neuroblastoma cell lines change their neurotransmitter phenotype from noradrenergic to cholinergic under retinoic acid treatment. Such "neurotransmitter switch" seems to be a consequence of changes in the expression and activity of the biosynthetic machinery for both neurotransmitters. In this study, we have characterized this "neurotransmitter switch" induced by retinoic acid in a human neuroblastoma cell line (NB69) showing catecholaminergic characteristics. Retinoic acid treatment reduced tyrosine hydroxylase activity and noradrenaline levels in NB69 cells but did not modify the expression of this enzyme. Moreover, the calcium-dependent release of [(3)H]noradrenaline in control cells was highly reduced by retinoic acid treatment. On the other hand, NB69 cells treated with retinoic acid enhanced the expression of choline acetyltransferase and acquired the capability to release [(3)H]acetylcholine in a calcium-dependent way. In addition, we found that the expression of the vesicular monoamine transporter 2 (VMAT2) and the vesicular acetylcholine transporter (VAChT) was increased in those cells treated with retinoic acid. Immunostaining revealed that retinoic acid treatment changed the cellular distribution of both vesicular monoamine transporter 2 and vesicular acetylcholine transporter. In conclusion, retinoic acid induces a noradrenergic to cholinergic switch in NB69 cells by acting at several levels of the neurotransmitter phenotypic expression.