Fifteen polyanionic bis-aldonic acid amides were synthesized by condensation of the corresponding aldonic acids and alpha,omega-diamines and by subsequent sulfation of the intermediates. The compounds were completely sulfated and exhibited molecular weights between 1450 and 2600 daltons. The antithrombotic activity of these synthetic heparin analogs was evaluated in a rat jugular vein clamping induced thrombosis model. Several compounds pounds were potent antithrombotic substances comparable in potency to the low molecular weight heparin Fraxiparin. Some structure activity relationships on their pharmacologic action could be deduced. The antithrombotic activity of a representative agent was confirmed using the Wessler stasis model in rabbits. In the in vitro tests the bis-aldonic acid amides exhibited moderate to low anticoagulant effects depending on the assay used. In the activated partial thromboplastin time assay, the anticoagulant potency of these compounds was between 2- and 30-fold lower than that of heparin. The anti-factor Xa activity of the experimental substances was at least 50 times lower than that of heparin, whereas the compounds were devoid of anti-factor IIa activity when measured in an amidolytic assay. The anticoagulant effects of these agents were dependent on the length of the spacing and on the type of aldonic acid moieties. These bisaldonic acid amides represent a novel class of potent antithrombotic substances without any anti-factor IIa activities and only very low anti-factor Xa activities, suggesting that these antiprotease actions are not a prerequisite for the antithrombotic action of polyanionic substances.