There is an ongoing controversy concerning whether crosslinked gamma chains in fibrin are oriented "transversely" between fibril strands or "end-to-end" along fibril strands. From the latter viewpoint, Veklich et al. [Proc Natl Acad Sci (USA) 95: 1438, 1998] observed that fibrinogen fibrils that had been assembled on a fibrin fragment E template, cross-linked with factor XIIIa, and then dissociated in acetic acid solution, were aligned end-to-end. This led to the conclusion that crosslinked gamma chains in fibrin under physiological conditions were also aligned end-to-end. To assess its validity we studied the assembly and organization of fibrinogen molecules on a des AB-fibrin fragment E (E-des AB) or a des A-fibrin fragment E (E-des A) template. We evaluated the roles of E polymerization sites E(A) and E(B), and D association sites gammaXL, Da, Db, betaC and alphaC in this process. E(A):Da interactions caused fibrinogen: E "DED" complexes to form, and markedly enhanced the gamma chain crosslinking rates of fibrinogen or des alphaC-fibrinogen. Fibrinogen crosslinking without added fibrin E was slower, and that of des alphaC-fibrinogen was still slower. These events showed that although alphaC domains promote fibrinogen fibril assembly and crosslinking, they contribute little to increasing the E(A):Da-dependent crosslinking rate. Electron microscopic (STEM) images of E-des AB and fibrinogen plus factor XIIIa showed single-, double-, and multistranded fibrils with interstrand DED complexes aligned side-to-side. This alignment was due to betaC:betaC contacts resulting from D subdomain rearrangements initiated by the E(B):Db interactions, and also occurred in mixtures of des alphaC-fibrinogen with E-des AB. In contrast, a mixture of fibrinogen and E-des A plus XIIIa revealed double-stranded fibrils with interstrand DED complexes in a half-staggered arrangement, an alignment that we attribute to crosslinking of gammaXL sites bridging between fibrils strands. These and other features of E-des A-based fibrinogen fibrils, including interstrand gamma chain bridges and early and extensive lateral fibril strand associations concomitant with accelerated gamma chain crosslinking, indicate that crosslinking of fibrin fibril strands takes place preferentially on transversely positioned gamma chains.