Hesperidin is a biologically active flavanone glycoside occurring abundantly in citrus fruits. In the present study, effects of intestinal microflora on pharmacokinetics of hesperidin were investigated using a pseudo-germ-free rat model treated with antibiotics. After administration of hesperidin to rats, hesperetin, hesperetin glucuronides, and metabolites postulated to be eriodictyol, hemoeriodictyol, and their glucuronides were detected in urine while hesperetin glucuronide was predominantly found in plasma. The plasma concentration-time profile of hesperetin was compared between non-antibiotic-exposed and pseudo-germ-free rats administered this compound. The maximal concentration (C(max)) values of hesperetin in non-antibiotic-exposed and pseudo-germ-free rats were 0.58 and 0.20 μg/ml, respectively, and area under the curve (AUC) values were 6.3 and 2.8 μg-h/ml, respectively. Thus, systemic exposure as evidenced by AUC and C(max) was significantly higher in normal compared to pseudo-germ-free rats. Fecal β-glucosidase activities of non-antibiotic-exposed and pseudo-germ-free rats were 0.21 and 0.11 nmol/min/mg, while fecal α-rhamnosidase activities were 0.37 and 0.12 nmol/min/mg, respectively. The rate of hesperidin transformation to hesperetin was 6.9 and 2.9 nmol/min/g in fecal samples in non-antibiotic-exposed and pseudo-germ-free rats, respectively. Taken together, these results showed that pharmacokinetic differences between non-antibiotic-exposed and pseudo-germ-free rats may be attributed to differing hesperidin uptake, as well as alterations in metabolic activities of intestinal flora.