The effect of the isoflavonoid phytoestrogens daidzein, equol, and genistein on sex hormone-binding globulin (SHBG) levels, SHBG mRNA transcript levels, and SHBG gene methylation was studied in HepG2 cell cultures by fluoroimmunometric SHBG assay and Northern and Southern hybridizations, respectively. The effect of 17 beta-estradiol on these parameters was studied as a control. The metabolism of isoflavonoids in HepG2 cells was determined by isotope dilution gas chromatography-mass spectrometry, after ion-exchange chromatography. Daidzein and equol increased SHBG levels in parallel intracellularly and extracellularly, whereas genistein increased SHBG levels only within the cells, resembling thus the effect of 17 beta-estradiol. The difference may originate from the fact that genistein has more hydroxyl groups than daidzein and equol. The regulation of SHBG production by phytoestrogens appears to occur at the post-transcriptional level. Firstly, daidzein, equol, or genistein did not have a clear effect on the steady-state SHBG mRNA levels. Secondly, no effect on SHBG gene methylation was observed by genistein. The findings applied also to 17 beta-estradiol. However, as the SHBG gene was more methylated in SHBG-negative MCF-7 cells than in SHBG-positive HepG2 cells, DNA methylation may play a role in the tissue-specific activation of this gene. The metabolism of isoflavonoids in HepG2 cells yielded mainly unconjugated and sulfated compounds. Similar metabolism in hepatocytes in vivo might retain their biological activity in tissues responsive to estrogens.