The effect of polyamines on the activity of the mixed-function oxidase (MFO) system from human, rat and rabbit liver microsomes was investigated in detail. It was shown that polyamine (spermine) stimulates NADPH-dependent activity of the MFO system several-fold whatever the substrate (foreign drug or natural), not only with microsomes but also with the reconstituted system consisting of highly purified cytochrome P-450 (LM2 isozyme), cytochrome P-450 NADPH reductase and dilauroylphosphorylcholine. Stimulation (extent and concentration dependence) appeared to be dependent on a number of parameters such as ionic strength, pH, animal species and treatment, nature of the substrate, and was stereospecific (different effect on 6 beta-and 16 alpha-testosterone hydroxylation). Further, the spermine effect was evaluated on some elementary steps of the cytochrome P-450 reaction cycle, like substrate binding, P-450 reduction and second electron transfer. Finally, it was shown that the organic peroxide dependent activity was not stimulated by spermine with microsomes nor with the purified P-450 LM2 isozyme. On the basis of this study, it was concluded that the locus of polyamine action is cytochrome P-450 and that stimulation could result either from increased stability of the oxyferrous intermediate of P-450 or from an increased rate of second electron transfer from reductase to P-450.