The cytostatic drug acetyldinaline [ACD, CI-994, 4-acetylamine-N-(2-aminophenyl)-benzamide] shows an extreme antileukemic effect in the Brown Norway (BN) rate model for acute myelocytic leukemia (BNML) with only minor toxicity for normal pluripotent hemopoietic stem cells. So far, the mode of action is unknown. A resistant subline (BNML/ACD-R) was developed in vivo in the BNML model. Leukemic rats received repeated oral administrations of ACD. When the leukemia relapsed after initial remission-induction with ACD, the cells were transferred to new recipients which were again treated. In total, the animals received 247 oral administrations of ACD (33 x 2 mg/kg per day and 214 x 5 mg/kg per day) before full resistance was reached. The cell line was transferred 17 times in total. Treatment of the final resistant cell line with therapeutically highly active doses of 23.7 mg/kg per day and 11.85 mg/kg per day ACD for 5 days, that resulted in an increase of life span (ILS) of 57 and 18 days, respectively, when applied to the sensitive parent BNML line (BNML/S), resulted in only 10 and 3 days ILS, respectively. These results indicate that a significant degree of resistance has been achieved, which can be overcome partially by increasing the dose of ACD. Whether the development of a resistant subpopulation of the BNML is a result of acquired resistance or whether a naturally resistant subpopulation has been selected out after prolonged treatment with ACD remains to be established. The currently available resistant subline BNML/ACD-R now offers the possibility for further studies on the mechanism of action of ACD.