When factor XIIIa-mediated crosslinking of fibrin or fibrinogen occurs, reciprocal intermolecular isopeptide bonds form first between paired carboxy terminal gamma chain donor-acceptor sites in outer molecular D domains, resulting in gamma chain dimers. Their location in the fibrin polymer is not certain, but some evidence suggests they are situated at the outermost ends of the D domains of linearly aligned molecules comprising each strand of double-stranded fibrils (''DD-long''). Other experiments indicate that gamma chain bonds are located between D domains in opposing fibril strands (''transverse''). To distinguish between these possible arrangements, we evaluated the ultrastructure of fibrils and fibers found in factor XIIIa-fibrinogen crosslinking mixtures, based on this reasoning: if DD-long bonding occurs, single-stranded fibrils should result, whereas transverse positioning will result in double-stranded fibrils. Fibrils formed in partially crosslinked fibrinogen solutions consisted of true parallel strands, as discerned visually from scanning transmission electron microscopic images and confirmed by mass per unit length fibril measurements. Neighboring fibrinogen D domains in each fibril strand were aligned end-to-end and were in register with a fibrinogen E domain in the opposite strand, creating a half-staggered molecular arrangement with similar to 22.5-nm periodicity corresponding to half the length of fibrinogen. Ribbon-like fibrinogen fibers, like fibrils, displayed 22.5-nm periodicity, as expected from laterally associated double-stranded fibrils with D domains in register. Taken together, these results indicate that carboxy terminal gamma chain bonds are positioned transversely between strands and are represented by thin filamentous structures bridging the D domains of opposing fibril strands-it follows that the same gamma chain crosslink arrangement occurs in fibrin. (C) 1995 Academic Press, Inc.