The disordered accumulation of hyaluronan, a nonsulfated glycosaminoglycan, is a hallmark feature of the tissue remodeling observed in thyroid-associated ophthalmopathy (TAO). Orbital fibroblasts have been shown to exhibit substantial up-regulation of hyaluronan synthesis when activated with proinflammatory cytokines such as interleukin-1β (IL-1β). Recently, three members of the hyaluronan synthase (HAS) gene family were cloned. Here we report that IL-1β can dramatically and consistently induce in orbital fibroblasts the expression of HAS2 in the five orbital strains examined. HAS3 messenger ribonucleic acid (mRNA) was also detectable in all these strains by RT-PCR under both control and IL-1β-treated conditions. In contrast, HAS1 mRNA was detected by Northern blot analysis in only one of the strains treated with IL-1β, but in three of five strains examined by RT-PCR. These HAS inductions by the cytokine were time dependent and could be attenuated with dexamethasone and cycloheximide. They were accompanied by an increased incorporation of[ ³H]glucosamine into hyaluronan, and dexamethasone could attenuate induction of macromolecular synthesis as well. Our observations suggest that the cytokine-dependent induction of the HAS genes in orbital fibroblasts may be the molecular basis at least in part for the increased accumulation of hyaluronan, driven by immunocompetent cells, in orbital connective tissue and the extraocular muscles in TAO.