The affinities of Factor XIII (FXIII), Factor XIIIa (FXIIIa), and cellular transglutaminase (Tg) for fibrinogen (Fgn), fibrin (Fbn), and fibronectin (Fn) were compared using a solid-phase binding assay. Initial rates of binding were as follows: FXIII bound Fbn 3-fold more than Fgn. FXIII did not bind Fn till 20 min. Increasing the ligands concentrations and binding time, resulted in weak binding of FXIII to Fn. FXIIIa bound Fbn 2-fold more than Fgn and 28-fold more than Fn. Tg bound Fn approximately 130-fold more than either Fgn or Fbn. At equilibrium, the extent of binding was determined to be as follows: FXIII bound Fbn 3-15-fold more than Fgn and 8-fold more than Fn. FXIIIa bound Fgn and Fbn equally and 12-25-fold more than Fn. FXIIIa bound Fgn or Fbn 2-fold and 25-fold greater than FXIII-Fbn and FXIII-Fgn interactions, respectively. Tg bound about equally to Fgn and Fbn and 10-20-fold less than Fn. The Kds' for FXIIIa binding to Fn, Fgn, and Fbn were 100, 23, and 19 nM, respectively. The Kd for Tg binding to Fn was 6.5 nM. The binding hierarchies are: [Tg-Fn] > [FXIIIa-Fgn] = [FXIIIa-Fbn] > [FXIII-Fbn] > [FXIII-Fgn] = [FXIIIa-Fn] > [Tg-Fbn] = [Tg-Fgn] > [FXIII-Fn]. Such hierarchies could regulate the cross-linkings by FXIIIa and Tg during hemostasis, wound healing, and cell adhesion.