Exposures of Sprague-Dawley rats to the soy isoflavone, genistein, throughout the entire lifespan have produced a number of effects on reproductive tissues, immune function, neuroendocrine function and behavior. Our previous studies investigated pharmacokinetics and disposition of genistein during adult and fetal periods and this study describes the internal exposures of post-natal day 10 (PND10) rat pups due to lactational transfer of genistein. Conjugated and aglycone forms of genistein were measured by using LC/MS/MS in serum (PND10) and milk (PND7) from lactating dams consuming a genistein-fortified soy-free diet, and in serum from their pups at a time when milk was the only food source (PND10). This study shows that limited lactational transfer of genistein to rat pups occurs and that internal exposures to the active aglycone form of genistein are generally lower than those measured previously in the fetal period. These results suggest that developmental effects attributable to genistein exposure in our chronic and multi-generation studies are more likely to result from fetal exposures because of the higher levels of the active estrogenic aglycone form of genistein in utero, although the possibility of neonatal responses cannot be excluded.