OBJECTIVE To study the mechanisms of nongenomic effect of 17beta-estradiol on human spermatozoa. METHODS The intracellular calcium ([Ca2+]i) in the spermatozoa was measured by flow cytometry after the spermatozoa was treated with the inhibitors of trans-membrane signaling transduction pathways and impermeable 17beta-estradiol (E2-BSA). Western blot was used to detect the activation of the signal proteins after the spermatozoa was treated with 1 x 10(-6) mol/L E2-BSA and tamoxifen, an estrogen receptor inhibitor. RESULTS Adenylyl cyclase (AC) inhibitor SQ22536, phospholipase C (PLC) inhibitor U73122 and protein tyrosine kinase (TPK) inhibitor Genistein all deterred the increase of [Ca2+]i caused by E2-BSA. E2-BSA also increased the PLC protein and PKC protein significantly. Tamoxifen, an antagonist of estrogen receptor, did not inhibit the activation of PLC caused by E2-BSA. CONCLUSIONS The E2-BSA has an effect on human spermatozoa in a nongenomic pathway, possibly through the transmembrane signal transduction in relation to AC, PLC and TPK.