Beta-chemokine, regulated on activation and normally T-cell expressed and presumably secreted (RANTES), is present in both the male and female genital tract fluids where its levels increase in diseases related to infertility, such as endometriosis and male genital tract infections. beta-Chemokine receptors (CCR3 and CCR5) are expressed on freshly ejaculated human sperm cells and a sperm chemoattractant effect for RANTES has been reported. No information exists on other possible roles of RANTES on sperm functions involved in the fertilization process. In the present study, the exposure of sperm suspensions to high concentrations of the chemokine, comparable to those observed in inflammatory diseases, significantly decreased the stimulatory effect exerted by progesterone on sperm/oocyte fusion, evaluated by means of the hamster egg penetration test. Accordingly, a large proportion of spermatozoa preincubated under capacitating conditions with high concentrations of RANTES underwent a premature acrosome reaction (AR) that prevented subsequent progesterone-induced AR. Finally, sperm samples exposed to the same high levels of chemokine showed a significant increase in the intracellular levels of cAMP, which is involved in capacitation and AR dynamics. These results indicate a negative interference of high levels of RANTES on the sperm fertilizing ability, thereby suggesting a potential contribution of this chemokine to subfertility associated with endometriosis and genital tract inflammatory diseases.