The Heptest heparin assay has recently been introduced, and evaluated for the laboratory monitoring of patients receiving low molecular weight heparins (LMWH). The aim of the present study was to elucidate the relative role on the Heptest assay of the anti-factors Xa and IIa activities present in the three types of compounds that possess: 1. exclusively anti-Xa activity (LF1: LMWH fractions with MW ranging from 1,200 to 4,200 D.); 2. both anti-Xa and anti-IIa activities (LF2 with MW from 4,800 to 12,000 D.); 3. exclusive anti-IIa activity (Hirudin and Dermatan Sulfate). All compounds studied demonstrated dose-dependent activities in both amidolytic and clotting assays. The LF2 in contrast to the LF1, additionally enhanced the clotting times of Heptest. This enhancement was shown to be due to the anti-Factor IIa activity of the agents. Heptest does not exclusively reflect Anti-Xa activity since it is influenced by agents containing exclusive anti-IIa activity like Hirudin and Dermatan Sulfate. At low concentrations of LF2, Heptest measures predominantly the anti-factor Xa activity while at higher concentrations it is influenced by the combined activity of anti-factor Xa and anti-factor IIa. However, Heptest sensitivity to anti-factor IIa is significantly lower than for anti-Xa activity.