A cell line derived from a murine teratocarcinoma was cultured in growth medium supplemented with either 10% or 1% fetal bovine serum or was grown in chemically-defined medium buffered with either a combination of HEPES [4-(2-hydroxyethyl)-1-piperazine-N''-2-ethanesulfonic acid] and sodium bicarbonate or with bicarbonate alone. The sugar content of a particular glycoprotein secreted by these cells was markedly different in each case. No differences were noted in antigenicity or amino acid composition, including amino and carboxyl terminal amino acids. Differences in MW were attributable to the difference in carbohydrate content. The level of glycosylation of proteins in vitro apparently is dependent on the medium composition, including apparently unsuspected components such as buffers.