Initial exposure of cultured limb-bud cells (stage 23-24) to 5-bromo-2'-deoxyuridine (BrdU) irreversibly inhibits differentiation to cartilage under three different culture conditions. The inhibition of chondroitin sulfate synthesis is partially reversed by D-xylose in limb-bud cells after treatment with BrdU. The activities of four enzymes involved in chondroitin sulfate production were reduced in BrdU-treated cultures, but the magnitude of decrease was far less than the decrease in glycosaminoglycan synthesis. The slight increase in the turnover rate of sulfated glycosaminoglyeans in BrdU-treated mesenchyme was not sufficient to account for the marked decrease in chondroitin sulfate content. The results suggest that BrdU treatment interferes with normal synthesis of chondroitin sulfate core protein in cultured limb-bud cells, bitt does not greatly diminish enzyme activities or UDP-sugar levels necessary for production of polysaccbaride chains.Culture of several types of differentiated cells in the presence of the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU) results in decreased synthesis of macromolecules characteristic of cell specialization (1-4). Differentiated cell expression is resumed upon removal of BrdU. In contrast to this reversible effect, BrdU irreversibly inhibits differentiation of certain precursor cell populations to specific phenotypes (5, 6). Irreversibility apparently depends upon treatment of cells at stages of development before "determination" has been stabilized. Differentiated chondrocytes are characterized by synthesis of large quantities of chondroitin sulfate proteoglycan. Production of chondroitin sulfate is sharply decreased in both differentiated chick chondrocytes growing in the presence of BrdU and cultures of chick limb-bud cells (stages 23-24) (7) initially exposed to BrdU then grown in its absence for 1A4 week&(5). Brett and Robinson (8) Glycosaminoglycan Synthesis and Turnover. On the ninth day of growth, cultures were labeled with 5 uCi of H235SO4,10 MACi of [3H]acetate, or 5 ,uCi of [I4C]acetate per ml of medium for 6 hr. For analysis of total glycosaminoglycan synthesis, cells and media were pooled and total glycosaminoglycans were isolated as described (9). For turnover measurements, fresh medium was added after a 6-hr pulse with 5 MCi of H2'5SO4 per ml on the ninth day of culture. Glycosaminoglycans were purified from cells plus fresh medium in 9-day, 11-day, and 13-day cultures, and radioactivity was determined by liquid scintillation counting.Enzyme Assays. Cells were scraped from culture dishes, washed twice with the appropriate buffer, and disrupted by 25 1-sec bursts with -a Branson Sonifier at 70 W. Lysis and homogenization were monitored with trypan blue stain. Xylosyltransferase (10) and N-acetylgalactosamine transferase assays (11) were performed on 10,000 X g supernatant