In previous studies, we reported the isolation and characterization of a Chinese hamster ovary cell mutant (pgsG) defective in glucuronyltransferase I (GlcATI). This enzyme adds the terminal GlcA residue in the core protein-linkage tetrasaccharide (GlcAb1,3Galb1,3Galb1,4Xylb-O-) on which glycosaminoglycan assembly occurs (Bai, X, et al, [1999] J. Biol. Chem. 274:13017-13024; Wei, G. et al. [1999] J. Biol. Chem. 274:7857-7864). Here, we show that incorporation of 35SO4 into glycosaminoglycans in the mutant is temperature-sensitive, with greater synthesis occurring at 33 oC compared to 37 oC. Wild-type cells show the opposite thermal dependence. Rabbit antiserum to hamster GlcATI failed to detect cross-reactive material in pgsG cells by immunofluorescence and Western blotting. Furthermore, expression of chimeric proteins composed of mutant GlcATI fused to IgG binding domain of protein A or to GFP did not yield the proteins at the expected mass. The GFP-tagged version appeared as a truncated protein, and immunofluorescence showed large perinuclear bodies at 30 oC. At 37 oC, the fusion protein was not readily detectable. Sequencing cDNAs from mutant and wild-type cells revealed a single base transition (G331A) in the open reading frame in pgsG cells, which resulted in a Val111Met substitution. These data suggest that pgsG cells contain a labile form of GlcATI that causes conditional expression of glycosaminoglycans dependent on temperature.