The present work deals with localization of previously unknown polymerization sites of the fibrin DD-fragment. D-dimer we obtained has a pronounced inhibitory effect on fibrin polymerization (IC50 = 0.06 μM). The inhibitory effect of the D-fragment disappeared after reduction and carboxymethylation. However, polypeptide chains βDD (Bβ134-461) and γγDD (γ63-411)2 of the DD-fragment, isolated by preparative electrophoresis, displayed their inhibitory activity. For instance, the rates of fibrin protofibril lateral association were decreased twice in the presence of βDD and γγDD chains at their molar ratios to fibrin of 0.40 and 0.15, respectively. The IC50 values for βDD and γγDD were 0.24 and 0.10 μM, respectively. Highly specific inhibition of protofibril lateral association suggests that the protofibril lateral association sites are located in Bβ134-461 and γ63-411 regions of the fibrin D-domain. Our data confirm those reported by Doolittle et al. regarding the γ-chain and a hypothesis about β-chain of fibrin D-domain (Yang, Z., Mochalkin, I., and Doolittle, R. F. (2000) Biochemistry, 97, 14156-14161).