This study was done to document further the mechanism of the antithrombotic effect of CY 216 after subcutaneous injection in the rabbit. We first measured the circulating anti-factor Xa and anti-thrombin activities expressed in either International Unit or Standard Independent Unit and from these activities we calculated the above critical length material (ACLM) and below critical length material (BCLM) levels. The clearance of the BCLM was half that of the ACLM. Then we determined the inhibitory effect of CY 216 on thrombin generation (TG) in platelet-poor plasma (PPP) and in whole blood. TG was both inhibited and delayed in whole blood, while it was only inhibited in PPP. The IC50 on TG in PPP and in whole blood were 1.80 × 0.16 and 4.33 × 1.01 $mUg.ml-1 respectively. After the injection, the inhibition of TG was significant as long as BCLM was detectable. The duration of the antithrombotic effect was essentially correlated to the ACLM level in the Wessler-thromboplastin model and to the BCLM level in the Wessler-serum model. Taken together, these results demonstrate that both ACLM and BCLM components of CY 216 are involved in its anticoagulant effect ex vivo as well as in its antithrombotic activity in vivo, and that the relative contribution of BCLM increases with the time after administration.