Previous studies have shown that D-xylose partially overcomes the puromycin inhibition of chondroitin sulfate synthesis in cultured chick embryo chondrocytes. Likewise, D-xylose stimulates chondroitin sulfate synthesis by limb bud mesenchyme cells previously treated with BrdU or limb bud cartilage cells treated with puromycin. The studies reported here show that p-nitrophenyl-beta-D-xylopyranoside and 4-methyl-umbelliferyl-beta-D-xylopyranoside cause a much greater stimulation than does D-xylose and are active at much lower concentrations. In contrast to D-xylose, the xylosides strikingly stimulate chondroitin sulfate synthesis in predifferentiated mesenchyme cells. The xylosides stimulate synthesis of chondroitin sulfate by rat glial cell tumor cells (RC-6), a mouse neuroblastoma (C1300, NB41A), and two strains of cultured rat hepatoma cells (HTC, H(4)). These results indicate that certain types of nonconnective tissue cells contain the enzymic machinery for synthesis of chondroitin sulfate which is normally not utilized because of limited synthesis of core protein and/or xylosyltransferase. The beta-xylosides may be used as a probe of the capacity of various cell types to synthesize sulfated glycosaminoglycans.