Fibronectin exists in a soluble form in plasma and lymph as well as in a relatively insoluble form in tissues. The disappearance of endogenously labeled fibronectin from plasma and its subsequent extravascular localization was studied over a 38-h period in normal rats (350-400 g) utilizing plasma fibronectin labeled in vivo with 75Se-selenomethionine. For comparative analysis, 125I-albumin was utilized in these dual isotopic experiments. After the simultaneous injection of 75Se plasma fibronectin (20 nCi) and 125I-albumin (2 microCi), all measured tissues demonstrated accumulation of both radiolabeled proteins in extravascular sites. Plasma fibronectin demonstrated a rather specific and high affinity for liver and spleen, which are enriched with reticuloendothelial cells. Albumin manifested the expected washout from extravascular sites, whereas fibronectin primarily displayed retention in tissues. The plasma disappearance of 75Se-labeled fibronectin was well described by two exponentials: an early phase with a half time of 0.52 h and a later phase with a half time of 21 h. To account for tissue retention of labeled fibronectin, a two-compartment kinetics model was required that included loss from the extravascular exchangeable compartment. Analysis of the disappearance kinetics and fibronectin distribution allowed estimation of the fractional turnover rate, pool size, and mean residence time. Accordingly the total rate of loss for plasma fibronectin was 0.51-0.54 mg/h. The calculated pool size of the soluble form of fibronectin was 15.5-16.3 mg. The mean residence time for exchangeable fibronectin was 29.6-29.9 h. These findings suggest that plasma fibronectin can be incorporated within the insoluble pool of fibronectin in tissues.