Daunorubicin (DNR) is a major front-line drug in the treatment of childhood acute lymphoblastic leukaemia (ALL). Previously, we showed that in vitro resistance to DNR at diagnosis is related to a poor long-term clinical outcome in childhood ALL and that relapsed ALL samples are more resistant to DNR than untreated ALL samples. In cell line studies, idarubicin (IDR), aclarubicin (ACR) and mitoxantrone (MIT) showed a (partial) lack of cross-resistance to the conventional anthracyclines DNR and doxorubicin (DOX), but clinical studies in childhood ALL have been inconclusive about the suggested lack of cross-resistance. In the present study we determined the in vitro cross-resistance pattern between DNR, DOX, IDR, ACR and MIT in 48 untreated and 39 relapsed samples from children with ALL using the MTT assay. The relapsed ALL group was about twice as resistant to DNR, DOX, IDR, ACR and MTT as the untreated ALL group. Thus, resistance developed to all five drugs. We found a significant cross-resistance between DNR, DOX, IDR, ACR and MIT, although in some individual cases in vitro anthracycline cross-resistance was less pronounced. We conclude that IDR, ACR and MIT cannot circumvent in vitro resistance to DNR in childhood ALL. Clinical studies may still prove whether IDR, ACR or MIT has a more favourable toxicity profile than DNR.