Altered glycosylation has turned out to be a universal feature of cancer cells, and in many cases, to correlate with altered expression or localization of relevant glycosyltransferases. However, no such correlation exists between observed enzymatic changes and the expression of the oncofetal Thomsen-Friedenreich (T)-antigen, a core 1 (Gal-β1 → 3-GalNAc-ser/thr) carbohydrate structure. Here we report that T-antigen expression, instead, correlates with elevated Golgi pH in cancer cells. Firstly, using a Golgi-targeted green fluorescent protein (GT-EGFP) as a probe, we show that the medial/trans-Golgi pH (pHG) in a high proportion of breast (MCF-7) and colorectal (HT-29, SW-48) cancer cells is significantly more alkaline (pHG ≥ 6.75) than that of control cells (pHG 5.9–6.5). The pH gradient between the cytoplasm and the Golgi lumen is also markedly reduced in MCF-7 cells, suggesting a Golgi acidification defect. Secondly, we show that T-antigen expression is highly sensitive to changes in Golgi pH, as only a 0.2 pH unit increase was sufficient to increase T-antigen expression in control cells. Thirdly, we found that T-antigen expressing MCF-7 cells have 0.3 pH units more alkaline Golgi pH than non-expressing MCF-7 cells. Fourthly, in all cell types examined, we observed significant correlation between the number of T-antigen expressing cells and cells with a markedly elevated Golgi pH (pHG ≥ 6.75). Consistent with these observations in cultured cells, cells in solid tumors also heterogenously expressed the T-antigen. Thus, elevated Golgi pH appears to be directly linked to T-antigen expression in cancer cells, but it may also act as a more general factor for altered glycosylation in cancer by affecting the distribution of Golgi-localized glycosyltransferases. © 2006 Wiley-Liss, Inc.