The action of hyperthermia treatments on tumor promotion separated into a two-stage protocol has been investigated. 7,12-Dimethylbenz[a]anthracene (DMBA) initiated dorsal skin of female SENCAR mice was promoted with either H2O2 or 12-O-tetradecanoylphorbol-13-acetate (TPA) (4 applications, 2 times/week) as the first stage of promotion, followed by promotion with mezerein (28 applications, 2 times/week) as the second stage. Hyperthermia (44 degrees C, 30 min) treatment of the skin at the time of stage II promotion only (just before each mezerein application) suppressed 100% of papillomas when H2O2 was used as a first-stage promoter and 96% when TPA was used as the first stage, as compared to unheated control animals. The same hyperthermia treatment given only at stage I of promotion had similar results. Hyperthermia treatments just before stage I TPA promotion (4 treatments only) followed by mezerein as the second stage reduced papilloma formation by 92%. When H2O2 was used as the first stage promoter and again mezerein as the second, papilloma frequency was reduced by 74%, as compared to unheated controls. This antipromotion activity of hyperthermia could not be linked to an inhibition of skin protease activity. Although papilloma frequency was markedly suppressed by hyperthermia during stage I promotion only, carcinoma formation was not. A similar number of carcinomas appeared in the groups of mice receiving hyperthermia with either H2O2 or TPA as first-stage promoters, as in comparable groups receiving no hyperthermia. In contrast, when hyperthermia treatments were given during stage II promotion with mezerein (using either H2O2 or TPA as stage I promoters), carcinomas (as well as papillomas) were markedly reduced. The results suggest that DMBA initiation creates two types of promotion-dependent cells, a majority with relatively low progression probability and a minority with relatively high progression probability. The former require both stage I and II promotion while the latter require only stage II promotion to form tumors. Hyperthermia treatments given during stage II promotion protected against promotion and progression of both types of initiated cells, but similar treatments only during stage I did not protect against promotion and progression of the latter. Although promotion was required for expression, relative progression probability appeared linked to initiation and not promotion events. These findings suggest that hyperthermia treatment of persons exposed to tumor-promoting agents may reduce the risk of induced tumorigenesis.