Intracellular levels of 4'-O-tetrahydropyranyladriamycin (THP) and adriamycin (ADM) were measured by a fluorospectroscopic method, and the former was shown to be taken up by L5178Y cells much faster than ADM; the uptake velocity of THP at 1 μg/ml was calculated to be about 170 times faster than that of ADM. High performance liquid chromatography of cell extracts indicated that THP exists mainly in nuclei intact without hydrolysis. The effect of THP in inhibiting [³H]thymidine incorporation into DNA also indicated that THP taken up by cells rapidly went to nuclei and inhibited DNA synthesis. Fifty percent inhibition concentrations of THP or ADM on [³H]-thymidine incorporation during a 60-minute period, 15 minutes after the addition, were 0.1 μg/ ml and 4.2 μg/ml, respectively. Similar results were obtained when L1210 cells were used in place of the L5178Y cells.