Isoflavones have been implicated as health protective agents which are present in soybeans at mg per gram level. To characterize their bioavailability in humans and assess potential health protective effects from physiological achievable amounts of soy food consumption, a series of human feeding studies were conducted. When subjects received soymilk isoflavones at single dose a day or three dose a day with dosages ranging from 2.7 to 10.3 [mu]mol/kg body wt, isoflavone absorption and excretion of subjects were dose dependent with much greater bioavailability of daidzein than that of genistein. Overall, urinary recovery of daidzein and genistein were 18-40% and 9-19%, respectively, of ingested doses. Great variation was observed in fecal recovery of isoflavones among individual subjects. Recoveries ranged from <1% to 8% of ingested amounts. In vitro anaerobic incubation of isoflavones with human feces showed that intestinal half-life of daidzein and genistein may be as little as 7.5 h and 3.3 h respectively. In two subjects whose fecal isoflavone recovery was 10-20 times that in others, their 48-h urinary isoflavone recovery was one-fold greater, especially genistein, than that of subjects with smaller fecal isoflavone excretion. This suggested that gut bacteria possessing many important biotransformation enzymes play an important role in determining the pattern and magnitude of isoflavone absorption and excretion in women. Variation of protein and fat content of background diets did not affect isoflavone bioavailability as reflected in their urinary recoveries. These data suggested that ad libitum diet may be appropriate for short-term soy feeding study. Four soy foods, cooked soybean, texturized vegetable protein, tofu or tempeh fed to subjects at 3.5 [mu]mol isoflavones/kg body wt in single breakfast dose with various amounts of isoflavone glucoside conjugates and aglycones did not differ in their bioavailability in women. Greater daily urinary excretion and plasma concentration of isoflavone, especially daidzein, in male subjects than female subjects were observed after one-week soymilk feeding at 3.1 or 6.2 [mu]mol isoflavones/kg body wt. NK activities were not significantly different between males and females, post- and pre-soymilk feeding. In conclusion, substantial amount of soy isoflavones can be absorbed and bioavailable in humans depend on doses, gut microflora and sex, but not background diet selection and types of soy foods.