Abstract The glycosphingolipids isolated from the detergent-insoluble material (DIM) of whole cells as well as from a similar detergent-insoluble substrate attachment matrix (DISAM) have been investigated in comparison with the glycosphingolipids of whole cells. The proportion of glycolipids in the total lipid extract was enriched in the DISAM as well as DIM fractions as compared to whole cells. The ratio of ganglioside (GM 3) to neutral glycolipids was also higher in the DISAM fractions than in whole cells. The radioactivity incorporated into DISAM glycolipids of BHK cells, metabolically labeled with radioactive glucosamine, was greater in confluent cells than in sparsely growing cells; however, label incorporation into glycolipids of the DISAM fraction of BHKpy cells was 2–3-fold higher than that of confluent BHK cells, although the chemical quantity of GM 3 in whole cells was much lower in BHKpy cells than in BHK cells. In order to confirm the enhanced label in DISAM glycolipids of BHKpy cells by other procedures, the labeled cells were detached by EGTA, washed, and reattached on plates. The amount of label in DISAM glycolipids of the reattached matrix of BHKpy cells was much higher than that of BHK cells. Cell spreading and cell attachment on plastic plate were inhibited by inclusion of GM 3 in the medium. These data suggest that: ( i) glycolipids, particularly GM 3, at the cell attachment site have different metabolic activity from those of whole cells; the label in glycolipids goes preferentially into cell attachment sites, and may have some functional role in regulating cell attachment of BHK cells; ( ii) metabolic activity and turnover of GM 3 in cell attachment sites of confluent cells are higher than actively growing cells, yet those of transformed cells are much higher than any state of non-transformed cells.