Factor XIII-A (FXIII-A) is present in the cytosol of platelets, megakaryocytes, monocytes, osteoblasts, and macrophages and may be released from cells by a nonclassical pathway. We observed that plasma FXIII-A levels were unchanged in thrombocytopenic mice (Bcl-x(Plt20/Plt20) and Mpl(-/-)), which implicates nonclassical secretion from nucleated cells as the source of plasma FXIII-A. We, therefore, examined the intracellular targeting of FXIII-A in the THP-1 (monocyte/macrophage) cell line and in human monocyte-derived macrophages. Metabolic labeling of THP-1 cells did not show release of (35)S-FXIII-A either under basal conditions or when interleukin 1-beta was released in response to cell stress. However, immunofluorescence of THP-1 cells and primary macrophages showed that FXIII-A associated with podosomes and other structures adjacent to the plasma membrane, which also contain trans-Golgi network protein-46 and Golgi matrix protein-130 (GM130) but not the endoplasmic reticulum luminal protein, protein disulphide isomerase. Further, FXIII-A was present in GM130-positive intracellular vesicles that could mediate its transport, and in other contexts GM130 and its binding partner GRASP have been implicated in the delivery of nonclassically secreted proteins to the plasma membrane. Hence, this mechanism may precede FXIII-A release into the extracellular matrix from macrophages and its release into plasma from the cell type of origin.