We have developed a sensitive ligand blot assay to detect hyaluronan (HA) binding proteins in cell extracts using 125I-HA. Samples to be tested are electrophoresed using standard, nonreducing SDS-PAGE conditions, electro-transferred to nitrocellulose then blocked in buffer containing Tween 20. After incubation with 125I-HA the nitrocellulose is washed and HA-binding proteins are detected by autoradiography. This method was used to detect different HA-binding proteins in isolated rat liver cell preparations. Two HA-binding bands of 175 kD and 350 kD were detected in sinusoidal endothelial cells. Both bands were competed virtually 100% with nonlabeled HA. Using rat hepatocytes, the assay detected major bands at 85 kD and 180 kD. In addition, histones present in both cell types were readily detected in the low MW region. Thus, two different liver cell types show different HA-binding patterns. The blocking procedure is critical for successful renaturation of HA-binding activity, since substitution of BSA for Tween 20 did not result in detectable 125I-HA-binding. This ligand blot assay will be a powerful tool to detect HA-binding proteins in various other tissues and cell types.