Growth characteristics, polyamine levels, and distribution of cells in the cell cycle were determined for 9L rat brain tumor cells treated for various periods with 1 mM dicyclohexylamine sulfate (DCHA). Continuous treatment of cells with DCHA caused growth inhibition at 2 days of treatment. After 2 days of treatment the growth rate of cells increased to approximately the same rate as control cells, even though treatment was continuous. Levels of spermidine were depleted to less than 10% of control levels, spermine levels were essentially unchanged, and putrescine levels were elevated to more than 350% of control levels after 9L cells were treated with DCHA for 2 days. In contrast to results found for the polyamine biosynthesis inhibitor alpha-difluoromethylornithine, treatment of 9L cells with DCHA did not potentiate the cytotoxicity of 1,3-bis(2-chloroethyl)-1-nitrosourea. To mimic the effects on polyamine levels caused by treatment with DCHA, 9L cells were treated with 5 mM putrescine alone or with 5 mM putrescine and 1 mM DCHA after treatment with 1 mM alpha-difluoromethylornithine. Results of these experiments suggest that treatment with DCHA alone does not potentiate the cytotoxicity of 1,3-bis(2-chloroethyl)-1-nitrosourea because elevated levels of putrescine caused by treatment counteract the effects of decreased spermidine levels.