We have previously demonstrated that transforming growth factor-beta1 (TGF-beta1)-mediated fibroblast-myofibroblast differentiation is associated with accumulation of a hyaluronan (HA) pericellular coat. The current study demonstrates failure of fibroblast-myofibroblast differentiation associated with in vitro aging. This is associated with attenuation of numerous TGF-beta1-dependent responses, including HA synthesis and induction of the HA synthase enzyme HAS2 and the hyaladherin tumor necrosis factor-alpha-stimulated gene 6 (TSG-6), which led to an age-related defect in pericellular HA coat assembly. Inhibition of HAS2-dependent HA synthesis by gene silencing, removal of the HA coat by hyaluronidase digestion, or gene silencing of TSG-6 or cell surface receptor CD44 led to abrogation of TGF-beta1-dependent induction of alpha-smooth muscle actin in "young" cells. This result supports the importance of HAS2-dependent HA synthesis and the HA coat during phenotypic activation. Interleukin-1beta stimulation, however, failed to promote phenotypic conversion despite coat formation. A return to basal levels of HA synthesis in aged cells by HAS2 overexpression restored TGF-beta1-dependent induction of TSG-6 and pericellular HA coat assembly. However, this did not lead to the acquisition of a myofibroblast phenotype. Coordinated induction of HAS2 and TSG-6 facilitation of pericellular HA coat assembly is necessary for TGF-beta1-dependent activation of fibroblasts, and both components of this response are impaired with in vitro aging. In conclusion, the HA pericellular coat is integral but not sufficient to correct for the age-dependent defect in phenotypic conversion.