Sarcoma virus transformed rat kidney cells, NRK[MSV-MLV] cells, and their non-transformed counterparts, NRK cells, were repeatedly subcultured in medium supplemented with 1 mM 2-deoxy-D-glucose (dGlc). These dGlc-grown cells exhibited alterations in glycosylation of surface proteins. Gel filtration analyses of pronase-treated, trypsin-sensitive, surface fucopeptides indicated a reduction in the average size of the fucopeptides from both dGlc-grown NRK and NRK[MSV-MLV] cells. When the cells were grown in control medium the fucopeptides from the NRK[MSV-MLV] cells were larger than those from NRK cells, i.e. the fucopeptide difference observed in a wide range of transformed versus normal cells. The changes in the fucopeptides of dGlc-grown cells were greater for the NRK cells so that after growth in dGlc-supplemented medium there was an enhancement in the typical fucopeptide differences between normal and transformed cells, namely a larger difference in both size distribution and degree of sialylation. These dGlc-related alterations were readily reversed when the cells were transferred to control medium. The changes in glycosylation were compatible with continued cell replication although there was an increase in doubling time of the dGlc-grown cells, 6-7 h increase for NRK cells, 1-2 h increase for NRK[MSV-MLV] cells.