The secreted and cell surface high molecular weight glycoconjugates (HMG) generated by primary cultures of airway epithelial cells from cystic fibrosis (CF) patients are oversulfated. To determine whether this abnormality is maintained in transformed CF airway epithelial cells and whether differences in transport or intracellular accumulation of sulfate can explain this alteration, we assessed sulfate metabolism in paired CF and normal cell lines as well as primary cultures of CF and normal cells. Both 4-acetamido-4'-isothiocyanostilbene-2, 2'-disulfonic acid-inhibitable and-resistant [35 S] sulfate efflux and influx were identical for each pair of CF and normal cell lines. Furthermore, cell content of inorganic sulfate was not significantly different in CF and normal cells. However, compared with primary CF cells that oversulfate HMG transformed CF cells oversulfated cell surface HMG but not HMG released into culture medium. Our results suggest that plasma membrane sulfate transport is not altered in CF airway epithelial cells and the abnormal sulfation of HMG may be due to perturbation in intracellular sulfate activation or transfer of activated sulfate to HMG. The relationship of this abnormality to CF transmembrane conductance regulator mutations remains to be determined.