This study compared the cytotoxicities of bioprosthetic heart valve materials crosslinked by glutaraldehyde, stored in 4% formaldehyde or Hank's solution. Human embryonic pulmonary fibroblasts or L-929 cell culture in vitro were used. Cell proliferative inhibition index(CP II) was calculated for bioprosthetic heart valve materials using different store methods in different rinse periods(before, 10 days, 20 days, 30 days). The results demonstrate: (1) bioprosthetic heart valve materials stored in 4% formaldehyde or in Hank's solution both have significant cytotoxicity, and the longer the rinse time continues, the lower the cytotoxicity declines; (2) HEL cell is more sensitive than L-929 cell in detecting the cytotoxicity of toxic biomaterials but for weak toxic biomaterials the two cell lines are not significanth different; (3) formatldehyde solution increases the cytotoxicity of biomaterials stored in it, but the enhanced cytotoxicity can be easily relieved by rinse. The authors conclude that bioprosthetic heart valve materials have long term significant cytotoxicity and the biomaterial cytotoxicity test using human fibroblasts is more sensitive and precise than other tests.