The incorporation of plasma fibronectin (pFn) into the extracellular matrix (ECM) is believed to influence tissue integrity, wound repair, and vascular permeability. In vitro, matrix assembly of Fn requires the binding of soluble Fn to cell-associated matrix assembly sites. Alkylation of human pFn (HFn) with N-ethylmaleimide (NEM) prevents the initial binding of Fn to matrix assembly sites as well as its in vitro incorporation into the ECM as reflected by detergent-insoluble 125I-labeled Fn (pool II Fn). We determined the kinetics of Fn matrix incorporation in tissue and whether NEM treatment of rat pFn (NEM-RFn) would limit its in vivo incorporation into ECM by analysis of pool I [deoxycholate (DOC) soluble] and pool II (DOC insoluble) 125I-Fn in tissues after its intravenous injection into rats. After intravenous injection, tissue incorporation of normal rat 125I-pFn was especially intense in liver and spleen, in agreement with the large amount of endogenous Fn detected in the matrices of these organs. Tissue deposition of plasma-derived 125I-RFn in liver and spleen peaked by 4 h, with significant (P < 0.01) loss over 24 h, indicating turnover of matrix Fn. Tissue localization of normal 125I-RFn in liver, lung, spleen, heart, and intestine was greater (P < 0.05) than 125I-NEM-RFn at 4 h. Normal HFn, but not NEM-HFn, was incorporated into tissues and colocalized with endogenous Fn in the matrix. To identify the cells mediating the intense incorporation of pFn into liver ECM, we compared matrix assembly of 125I-HFn by cultured fibroblasts, hepatocytes, and hepatic Kupffer cells. With fibroblasts, 125I-HFn in pool I reached steady state by 3 h, whereas 125I-HFn in pool II exceeded that in pool I by 6 h and continued to increase over 24 h. With hepatocytes, pool I 125I-HFn reached steady state by 1 h, and a progressive increase (P < 0.05) of 125I-HFn in pool II was observed over 24 h. Kupffer cells were not able to incorporate significant amounts of 125I-HFn into matrix. NEM-HFn displayed limited incorporation into ECM by both fibroblast and hepatocyte cultures. These novel observations suggest that the interaction of soluble pFn with matrix assembly sites is necessary to its in vivo incorporation into the ECM.