Candida albicans is both a commensal and a pathogen in the oral mucosa. Previous studies have indicated that epithelial cell-associated carbohydrate moiety can inhibit C. albicans growth. In the present study, the mechanisms by which epithelial cells inhibit Candida growth were studied by examining the effect of hyaluronan (HA). A coculture of C. albicans and KB cells or COS-7 cells inhibited in vitro growth of the fungus by 50-87% at an effector-to-target (E:T) ratio of 80:1. Removing extracellular HA by hyaluronidase caused a significant decrease in the anti-Candida activity of the cells. In addition anti-Candida activity was observed at 1 micro g/ml HA (2000 kDa). The antifungal activity of extracellular HA was further studied by transiently transfecting COS-7 cells with human HSA1, HSA2, or HSA3 in order to produce high levels of extracellular HA. All of the transfectants inhibited C. albicans growth in vitro by 51-65% compared to 38% inhibition by the vector control (P<0.05). These results suggest that the anti-Candida activity of epithelial-cells is mediated by extracellular HA.