We investigated whether the in vivo growth inhibitory effect of the combination of 4-amidinoindan-1-one 2′-amidinohydrazone (CGP 48664A) and α-difluoromethylornithine (DFMO) is reversible by treatment with N 1-acetylspermine ( N 1-acSp). DBA-2 mice were inoculated with 10 5 L1210 cells i.p. on day 0. From day 1 they received 2.50 mg CGP 48664A/kg i.p. once daily and 500 mg DFMO/kg i.p. twice daily. On day 5 they received 3×2500 nmol N 1-acSp i.p. with 15-min intervals. L1210 cell numbers, S-phase percentage and polyamine contents, and liver and spleen polyamine contents were monitored in the following 48 h. Four days treatment with CGP 48664A/DFMO reduced L1210 cell numbers, S-phase, and spermidine. N 1-acSp treatment increased L1210 spermidine from ≤8 h and percentage S-phase from 12 h. Maxima for spermidine and S-phase were reached at ≤8 and 18 h, respectively. These were below levels of untreated controls. Decreases were noted from 12 and 18 h, respectively. N 1-acSp was detectable in L1210 from 0–18 h. Liver spermidine was decreased by CGP 48664A/DFMO. After N 1-acSp treatment, liver N 1-acSp and N 1-acSd increased from ≤8 h, reached maxima at ≤8 and 10 h, respectively, and were undetectable from 15 h. We conclude that the in vivo growth inhibitory effect of CGP 48664A/DFMO is reversible by N 1-acSp treatment. The liver is probably involved in N 1-acSp terminal catabolism. The effect of the polyamine depletion–repletion scheme on S-phase cell numbers may be much more profound than present estimates from 5-bromo-2′-deoxyuridine incorporation.