Hyaluronan (HA) is an essential component of synovial interstitial matrix and synovial fluid, but the link between its production and joint use is unclear. HA secretion is enhanced by joint distension in vivo, but direct proof that synoviocytes exhibit mechanosensitive HA secretion is lacking. We tested this in vitro. Primary rabbit synoviocyte (PRS) cultures from microdissected synovial intima were subjected to 180 min of maintained 10% static stretch, or to 10 min of 10% static stretch followed by 170 min relaxation, in a Flexcell 2000 apparatus. Stretch stimulated HA secretion into the medium over 3 h by 57%. Notably, a short stretch (10 min) was as effective as sustained stretch. Actinomycin D and cycloheximide abolished stretch-stimulated HA secretion and also reduced basal HA secretion rate. RT-PCR showed that HAS2 was the major hyaluronan synthase expressed, but there was no increase in HAS2 mRNA (or other isoforms) in continuously stretched cells, and only a small increase (20%) at 180 min in cells stretched for the first 10-30 min. However HAS2 transcription increased 10-fold in response to TGF-beta1 and IL-1beta. Thus HA secretion by intimal synoviocytes is regulated by a mechanosensitive pathway which depends on transcription and de novo protein synthesis, possibly of HAS2, but also of other proteins involved in HA secretion.