After fibrin polymerizes to form a clot, the transglutaminase Factor XIIIa cross-links the gamma and alpha chains to stabilize the clot. There has been conflicting evidence on whether the gamma chain isopeptide bonds occur between molecules that are interacting in a longitudinal (end-to-end) manner or transverse (half-staggered) manner between the two strands of the protofibril. Since the topology of the cross-links has important consequences for fibrin structure, as well as for its stability and susceptibility to and pattern of fibrinolysis, cross-linked fibrin fragments were examined by electron microscopy to distinguish between these two possibilities for the arrangement of the ligated molecules. Cross-linked fibrin clots were produced by prolonged incubation of fibrinogen with thrombin and Factor XIII, and then digested with plasmin. The resulting soluble cross-linked fibrin complexes were rotary-shadowed with tungsten and examined by electron microscopy, revealing protofibril-like structures consisting of clusters of globular domains with a repeat of 22.5 nm. Longer plasmin digestion times yielded increasingly shorter structures. Rotary-shadowed cross-linked fibrin fragments, produced by dilution of the complexes into 0.125% acetic acid at pH 3.5 to dissociate all non-covalently linked fragments, showed uniformly single-stranded structures with a characteristic spacing of nodules, consistent with longitudinal cross-linking. Long, thin strands were seen at short digestion times, while shorter strands appeared with longer digestion. The smallest structures observed included two nodules together, and two such nodules with another nodule at a short distance from one or both ends, compatible with fragments DD, DY, and YY. Longer strands had the appearance of fibrin molecules that were linked end-to-end, usually with a fragment D or Y at each end. In conclusion, these results are consistent with previously proposed structures of these derivatives and clearly demonstrate that the interactions between cross-linked gamma chains are longitudinal (end-to-end) and not transverse.