ABSTRACT—BCNU (carmustine), 5‐Fluorouracil (5‐FU) and Vidarabin‐mono‐phosphate (ARA‐A5'P) were compared in their activities against 30 cell lines of primary (n = 21) and metastatic (n = 9) brain tumors, which were characterized in tissue culture by cytochemical, immunological and cytogenetic criteria.In vivoachievable concentration‐time products were correlated within vitropharmacokinetic data. A micro assay was employed to screen for drug toxicity in individual tumor cell lines; cells were exposed to the drugs at exposure doses relevant toin vivopharmacokinetics. After 5–8 population doubling times of untreated controls, RNA‐synthesis, as a parameter of cell metabolism and proliferation, was determined by incorporation of (5, 6‐3H)‐uridine into cellular RNA (liquid scintillation counting protocol). A tumor stem cell assay was performed under similar conditions. The cytotoxic effect of each drug on individual cell lines was expressed in terms of a sensitivity index SI (SI = 1 indicating complete resistance) to compare effects of different drugs on the individual tumor cell lines. Mean sensitivity indices for ARA‐A5'P, BCNU and 5‐FU in brain tumor cell lines (in brackets: primary CNS‐tumors) were 0.64 (0.59), 0.89 (0.82) and 0.35 (0.33) respectively. 5‐FU was significantly more active than BCNU and ARA‐A5'P(P<0.001), whereas BCNU was significantly less active than ARA‐A5'P (P<0.001). ARA‐A5'P had a suppressive effect on formation of brain tumor stem cell colonies. There was no cross‐resistance of ARA‐A5'P to either BCNU or 5‐FU. We conclude that ARA‐A5'P and 5‐FU are potent agents in experimental ther