A new procedure is introduced for the isolation of connective tissue fibers, called biomatrix, containing a significant portion of the extracellular matrix (basement membrane components and components of the ground substance). Biomatrix isolated from normal rat liver contains >90% of the tissue's collagens and all of the known collagen types, including types I and III and basement membrane collagens. The purified collagenous fibers are associated with noncollagenous acidic proteins (including fibronectins and possibly small amounts of glycosaminoglycans). Procedures are also described for preparing tissue culture substrates with these fibers by either smearing tissue culture dishes with frozen sections or by shredding the biomatrix into small fibrils with a homogenizer. The biomatrix as a substrate has a remarkable ability to sustain normal rat hepatocytes long-term in culture. The hepatocytes, which on tissue culture plastic or on type I collagen gels do not survive more than a few weeks, have been maintained for more than 5 mo in vitro when cultured on biomatrix. These cells cultured on rat liver biomatrix show increased attachment and survival efficiencies, long-term survival (months) and retention of some hepatocyte-specific functions.