A manual chromogenic substrate method for the determination of heparin cofactor II (HC II) activity in plasma has been adapted to the Cobas Bio centrifugal analyzer. By reducing the dermatan sulfate concentration used, interference by opacity was avoided, and the correlation between the manual and the new automated method was high (r = 0.96). Plasma samples from 182 females and 197 males were examined. The mean values of HC II activities in this normal population was relatively high, with a total range of 43–150% and a standard deviation of 17%. 4 apparently healthy individuals had levels below 60%. In males, the mean HC II activity was higher in the 50–59 yr age group than in either the 20–29 yr group or in the group above 60 yr (p < 0.05). In females, the postmenopausal age group had higher mean HC II activities, but the difference was not statistically significant.