To get more insight into possible species differences in the bioactivation of estragole, the kinetics for sulfonation of the proximate carcinogen 1'-hydroxyestragole were compared for male rat, male mouse, and mixed gender human liver S9 homogenates. In order to quantify sulfonation, 2'-deoxyguanosine was added to the incubation mixture in which sulfonation of 1'-hydroxyestragole was catalyzed to trap the reactive 1'-sulfooxyestragole. A method was developed with which the formation of the most abundant adduct with 2'-deoxyguanosine could be quantified using isotope dilution LC-ESI-MS/MS. Comparing the kinetics for sulfonation by liver S9 homogenates of male rat, male mouse, and humans revealed that sulfonation was about 30 times more efficient by male rat liver S9 than by human liver S9, whereas the catalytic efficiency by male mouse and human liver S9 was about the same. This indicates, as far as the bioactivation by sulfotransferase is concerned, that when extrapolating the cancer risk from laboratory animals to humans, using data from male rats may overestimate the cancer risk in humans, whereas using data from male mice may provide a better estimate of the cancer risk in humans.