Data from epidemiological and human intervention studies have highlighted potential cardiovascular benefits of soy isoflavone-containing foods. In humans, genistein and daidzein are extensively metabolized after absorption into glucuronides and sulfate metabolites. However, limited data exist on isoflavone cellular metabolism, in particular in endothelial cells. We investigated the uptake and cellular metabolism of genistein, daidzein and its major in vivo microbial metabolite, equol, in human endothelial (HUVEC), liver (HepG2) and intestinal epithelial cells (Caco-2 monolayer). Our results indicate that genistein and daidzein are taken up by endothelial cells, and metabolized into methoxy-genistein-glucuronides, methoxy-genistein-sulfates and methoxy-daidzein-glucuronides. In contrast, equol was taken up but not metabolized. In HepG2 and Caco-2 cells, glucuronide and sulfate conjugates of genistein and daidzein and a sulfate conjugate of equol were formed. Our findings suggest that endothelial cell metabolism needs to be taken into account when investigating the cardioprotective mechanisms of action of isoflavones.