Glycosaminoglycan (GAG) synthesis was studied in untransformed and transformed normal and Werner syndrome (WS) fibroblasts, because WS manifests pleiotropic abnormalities in connective tissue. Continuous labelling of cells with [3H] glucosamine and [35S] sulfate for 48 hours revealed enhanced synthesis of cellular GAG, more rapid transfer of these into the pericellular fraction, and more accumulation of GAG in the medium in cultures of untransformed WS fibroblasts compared with cultures of normal diploid cells. Total GAG in the 24 hour medium from confluent cultures was composed of 80-90% hyaluronic acid (HA) and 10-20% sulfated GAG (S-GAGs) in both untransformed normal and WS fibroblasts, whereas it was approximately 50% each HA and S-GAG in transformed normal and WS cells. The proportional enhancement of [35S] GAG synthesis in response to exogenous beta-D-xylopyranosides was similar in normal and WS cells, although transformed cells demonstrated only approximately one-half the enhancement observed in non-transformed cells. Thus, the overall activity of GAG synthesis is not grossly altered by the WS gene mutation. Enhanced synthesis and accumulation of HA and dermatan sulfate (DS) in the medium was characteristic of untransformed WS fibroblasts, but appeared to be normalized in an SV40-transformed WS cell line (PSV811), as in transformed normal cells (WI38CT-1). We need more experiments to determine whether aberrant GAG metabolism in WS cells is a direct or indirect expression of the primary gene defect.