Time relationships of drug concentrations in tissue of a transplantable rat rhabdomyosarcoma and of tumour responses up to I20 hr after treatment with methotrexate (MTX) were analysed and compared. MTX was shown to be retained within the tumour in a substantial concentration for several days, although no evidence of MTX polyglutamation was obtained. The response data confirm that MTX is active in the tumour for up to at least 3 days after injection. Within the first day after MTX treatment the nucleotide pools are only partly depleted. This indicates that the inhibition of DNA synthesis is still incomplete at the time when salvage precursors in increasing amounts are becoming available from decaying cells. From flow cytometric analysis of cell-cycle progression it is concluded that subsequent cohorts arriving in early S-phase were retarded, but not inhibited, in their progression through the S phase. At 3 days after MTX treatment the mean rate of cell-cycle progression as well as the relative clonogenic capacity were maximally reduced to 30% and I 'Yo of control values, respectively. From 3 to 5 days the rate of cell-cycle progression was gradually restored, whereas from day 5 onwards the clonogenic capacity