Neuroblastomas in culture are characterized by the presence of 2 morphologically and biochemically distinct phenotypes (i.e., neural "N-type" and flat substrate-adherent "S-type") which undergo transdifferentiation. Human neuroblastoma SK-N-BE(2) cells differentiate toward a neural phenotype upon retinoic acid (RA) treatment. However, we recently showed that, during the RA treatment, a subset of SK-N-BE(2) cells undergo apoptosis; these cells specifically express a high "tissue" transglutaminase (tTG) level. This study was undertaken to investigate the cellular and molecular basis of the action of retinoic acid on apoptosis in human neuroblastoma cells. As a biochemical marker of the phenomenon we studied the tTG gene expression in the parental line SK-N-BE(2) and in 2 clones which stably express neuroblastic [BE(2)-M17] and substrate-adherent [BE(2)-C] features, respectively. Data showed a differential phenotype-specific regulation of tTG gene expression. In fact, RA treatment enhanced tTG expression and apoptotic index in the flat substrate-adherent variant, whereas, in cells expressing the neural phenotype, very low tTG expression and apoptosis were found. Northern-blotting analysis revealed that the substrate-adherent cells had a basal 3-fold higher level of tTG mRNA. An increase in tTG mRNA major transcript levels (3.7 kb) occurred within a few hours of exposure to RA in both the phenotypic variants. By contrast, tTG protein level was very low in the cell expressing the neuronal phenotype, even after prolonged exposure to RA. Immunohistochemical analysis indicated that tTG protein, in addition to mature apoptotic cells, was specifically localized in the flat substrate-adherent variant both in the wild-type and in the BE(2)-C clone. These findings suggest that the ability to undergo apoptosis in the neuroblastoma cells is associated with the expression of a non-neuronal neuroectodermal (substrate-adherent cells) immature phenotype.