Previous studies of the binding properties of fibronectin (Fn) have utilized methods whereby one or the other macromolecule was immobilized on a solid phase. In order to examine the interaction between human plasma Fn and gelatin in solution, the latter was labeled with fluorescein isothiocyanate (FITC) whose fluorescence polarization (P) served as a sensitive indicator of the formation of soluble complexes. Changes in P were detectable at Fn concentrations below 10(-9) M and continued up to concentrations above 10(-6) M at pH 7.3 and 25 degrees C. Fractionation of FITC-gelatin by exclusion chromatography and titration of selected fractions revealed a trend towards higher affinity with increasing size. A high-molecular-weight fraction comprised of beta and gamma components and a low-molecular-weight fraction comprised primarily of alpha chains exhibited sigmoidal increases in P (apparent positive cooperativity) with 50% saturation near 10(-9) and 10(-8) M Fn, respectively. By contrast, a 42-kDa chymotrypsin-generated Fn fragment which retains the ability to adhere to gelatin-Sepharose exhibited normal (noncooperative) binding to both gelatin fractions with Kd = 7 X 10(-7) M. In all cases, the increase in P could be reversed by addition of excess unlabeled gelatin or urea. The interaction of FN with FITC-gelatin provides the basis for a fast and sensitive determination of Fn levels in plasma and other fluids. Interference caused by other proteins such as albumin, which has an affinity for the fluorescein moiety, could be minimized by addition of 1.0 M NaCl which had no effect on the interaction between Fn and gelatin.