Assay conditions for determining hyaluronic acid levels in cultured cells have been examined. In cultures labeled with [3H]glucosamine, hyaluronic acid is measured by digestion with a highly specific hyaluronidase from Streptomyces hyaluronlyticus. Products obtained in the presence and absence of preliminary enzyme digestion are precipitated with cetylpyridinium chloride. The precipitation step has been optimized for ion concentration, glycosaminoglycan carrier and for cetylpyridinium chloride levels. Chondroitin sulfate is an effective carrier in the precipitation of radiolabeled product, while unlabeled hyaluronic acid is not. Addition of sulfate to the mixture yields a flocculent precipitate that facilitates subsequent steps of the determination. Optimizing these steps in hyaluronic acid determination can generate two- to three-fold increases in apparent levels of deposition in cultured cells.