Previous studies on mucous glycoprotein secretion in respiratory epithelium most often provided qualitative, rather than quantitative, data. This study describes a new technique to measure the secretion rate, pool size, and turnover time of the pool of sulfated mucous glycoproteins of dog tracheal epithelium. The technique involved interposing dissected tracheal epithelium between the halves of an Ussing-type chamber, incubating the submucosal side of the tissue with 35SO4, and measuring the rate of appearance of nondialyzable 35SO4 on the luminal side of the chamber of both during the labeling and "washout" of the mucous pool. By analyzing the pattern of elimination, we showed that 1) a steady secretion rate of labeled mucous glycoprotein occurs within 3-4 h when free 35 SO4 is present on te submucosal side of the tissue, 2) secretion is consistent with first-order kinetics from a single pool, 3) puromycin decreases the rate of secretion of labeled mucous glycoprotein, 4) secretion rate is greater in medium 199 than in modified Krebs-Henseleit solution, and 5) 1.2 mM SO4 supports maximal baseline secretion. In six tracheas, bathed in Krebs-Henseleit solution, the secretion rate was (mean +/- SE) 467 +/- 74 pmol SO4 x cm-2 x h-1, pool size, 964 +/- 144 pmol SO4/cm2, and turnover time, 2.12 +/- 0.16 h. This technique provides a quantitative method to characterize kinetics of sulfated mucous glycoprotein secretion.