Carbon dioxide is a metabolic byproduct of mammalian cell metabolism that can accumulate in poorly ventilated cultures. A buildup of CO2 at constant pH will be accompanied by an increase in medium osmolality. We have examined the glycosylation of tissue plasminogen activator (tPA) produced under serum-free conditions by recombinant Chinese hamster ovary (CHO) cells (MT2-1–8 cell line) in response to elevated pCO2 at constant or elevated osmolality. The proportion of sialic acids comprising N-glycolylneuraminic acid decreased from 2.3–4.0% under 36 mmHg pCO2 to 1.5–2.2% under 250 mmHg pCO2. No changes were observed in the total sialic acid content, the content of other monosaccharides, the relative amounts of type I and type II tPAs, the distribution of surface charges, or the proportion of high-mannose oligosaccharides—even though these conditions have previously been shown to inhibit the specific growth rate of MT2-1–8 cells by 30–40% and the specific tPA production rate by as much as 40%. These results suggest robust glycosylation of tPA by CHO cells.