Heparin is a universal drug used frequently for its anticoagulant effects. The variabilities in distribution and tendency of heparin to accumulate in tissues cause increased tissue concentrations despite normal serum levels. We aimed to underline the toxic effects of heparin in cell culture make projections for clinical applications. L929 mouse fibroblastic cell line was plated in 96-well culture plates at an initial density of 5000cells/well. Heparin was prepared in 10 different concentrations (10-300units/well). Following 3 days of incubation, viabilities were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for each concentration in each day and compared. The viability of cells decreased significantly with increasing doses of heparin; at least 50units/well in the first and second days and at least 20units/well in the third day (P<0.05 for each). There was statistically significant difference when the viabilities of cells treated with same heparin concentration in different days were compared (P<0.05). The authors clearly demonstrated the toxic effects of heparin in cell culture, toxic effects increased as the dose increased. To prevent the unwanted clinical side-effects of heparin further studies should be made and more accurate testing methods should be developed to determine the effective tissue concentration of heparin. (C) 2013 Wolters Kluwer Health | Lippincott Williams & Wilkins.