Differentiation therapy with retinoic acid has been considered a potential approach for treating rhabdomyosarcoma. Analysis of retinoids as differentiating agents for rhabdomyosarcoma is, however, rendered incomplete by the fact that some rhabdomyosarcoma cell lines are retinoic acid resistant. Therefore, the aim of the present work was to study the effect of all-trans-retinoic acid on two rat tumour cell lines, derived from the same rhabdomyosarcoma tumour model (i.e. the moderately differentiated low metastatic F21 cell line and the poorly differentiated high metastatic S4MH cell line), to discover how degree of differentiation and glutathione metabolism influence response to this retinoic acid derivative. We observed that whereas in the S4MH cell line all-trans-retinoic acid induced a significant inhibition of tumorigenic potential, in F21 cells all-trans-retinoic acid enhanced tumour growth and only at a higher dose was there a slight antiproliferative effect. These effects were in consonance with the activity level of gamma-glutamyltranspeptidase, which was significantly increased in F21 cells, but not in S4MH cells, in response to the all-trans-retinoic acid-induced increase in reactive oxygen species. The pro-tumour effect observed in F21 cells was reversed by adding buthionine sulphoximide, a specific cellular glutathione-depleting agent, to the all-trans-retinoic acid treatment. This combination produced a decrease in gamma-glutamyltranspeptidase activity, and an increase in oxidative stress and apoptosis. Our findings suggest that the response to all-trans-retinoic-acid of the tumour cell lines studied is influenced by the strong relationship between intracellular glutathione content, gamma-glutamyltranspeptidase activity and degree of differentiation of the rhabdomyosarcoma cell line, and that this relationship should be taken into account when identifying 'retinoid-sensitive' tumours.