Antithrombin III (AT-III) was determined functionally using chromogenic substrate (S-2238) and immunologically using radial immunodiffusion (RID) in plasma and serum from 115 blood donors. There was a decrease in functional activity by 43.5% and in antigen concentration by 18.4% during in vitro coagulation when corresponding serum and plasma samples were compared. A positive correlation was found between the two methods in plasma (r = 0.784) as well as in serum ( r = 0.658). RID in serum correlated well with RID in plasma (r = 0.811), but the correlation for S-2238 in plasma and serum was poor (r = 0.411). Functional AT-III decrease during coagulation was uncorrelated with age and fibrinogen, and was not statistically affected by sex, smoking or blood groups. Preoperative functional AT-III activity was measured in 25 patients undergoing elective hip replacement, 60% of whom developed postoperative thrombosis. The functional activity in serum discriminated well between the thrombotic and nonthrombotic group of patients (p less than 0.025), whereas the activity in plasma showed only a minor difference.