The relationship between the structure of ketonucleosides and cytotoxicity was investigated in Friend leukemia cells (FLC). When cells were grown in the continuous presence of ketonucleosides, it was shown that the addition of an electrophilic agent (Br-) to the sugar moiety (compound KN-35) increased the cytotoxic potential by ten fold as compared to the unsubstitute compound (KN-43). In contrast, addition of 0-acetyl group (compound KN-3) reduced this effect by three fold. When cells were pre-treated with KN-35, followed by growth in drug-free medium, cell survival was inhibited by 50% (ID50) after 3 min, whereas the same effect was reached after 240 min pre-treatment with KN-43. When drugs were pre-incubated in serum-free medium prior to cell exposure, reduced cytotoxicity was observed. We therefore conclude that the activity of these ketonucleosides may be related to the rate of in fact drug incorporation.