beta-D-Xylosides have been used to perturb proteoglycan (PG) synthesis to elucidate the function of PGs in a number of cellular processes, including proliferation, migration, and differentiation. This study was designed to examine whether specific xylosides affect the proliferation of several different cell types and, if so, whether this effect is dependent on altered PG synthesis via the false acceptor pathway. Both methylumbelliferyl beta-D-xylopyranoside and p-nitrophenyl beta-D-xylopyranoside (PNP beta-xyloside) inhibit cell proliferation and modulate PG synthesis; however, the alpha form of PNP xyloside which does not perturb PG synthesis inhibits the proliferation of cultured cells on a molar basis equally as well as the beta form. Conversely, beta-methyl xylopyranoside stimulates the synthesis of free glycosaminoglycan chains equally as well as PNP beta-xyloside and yet has no measurable effect on cell proliferation at comparable doses, indicating that cells can grow normally while experiencing disruption of their proteoglycan metabolism. At doses ranging from 0.5 to 5 mM, PNP beta-xyloside arrests cells in the G1 phase of the cell cycle at the same time point as serum starvation. It also delays the exist of cycling cells from the S phase. This treatment is not cytotoxic and is rapidly reversed by the replacement of PNP beta-xyloside containing medium with control medium. Dimethyl sulfoxide, the most commonly used solvent for beta-xyloside in proteoglycan studies, potentiates the inhibitory effect of PNP beta-xyloside on cell proliferation. These results indicate that the perturbation of PG synthesis via the false acceptor pathway can be uncoupled from control of cell proliferation.