Glycosaminoglycans (GAG) and proteoglycans, which are components of the extracellular bone matrix, are also localized in and at the membrane of osteoblasts and in the pericellular matrix. Due to their interaction with several growth factors, water and cations these molecules play an important role in regulating proliferation and differentiation of osteoblasts and bone development. The aim of this study was to assess in vitro the effects of two chemically sulfated hyaluronan (HyaS) derivatives on the proliferation of rat calvarial osteoblasts and to compare with those of native hyaluronan (Hya) and natural sulfated GAG such as chondroitin-4-sulfate (C4S), chondroitin-6-sulfate (C6S), dermatan sulfate (DS) and heparan sulfate (HS). Moderately and highly sulfated HyaS derivatives caused a time-dependent reduction of osteoblast proliferation. The anti-proliferative effect of HyaS was accompanied by a cell cycle arrest in the G1 phase, but was not associated with cell death. Whereas non-sulfated high molecular weight (HMW)- and low molecular weight (LMW)-Hya as well as C4S, C6S, DS and HS showed no effect on the cell proliferation.