We studied the metabolism of sulfated cell-surface macromolecules in dog tracheal epithelial cells in primary culture. To examine the time-course and rate of appearance of sulfated macromolecules at the cell surface, the cells were pulsed with 35SO4 for short periods (5-15 min), and the incubation medium was sampled for spontaneously released macromolecules (basal secretions) and for release induced by trypsin (trypsin-accessible secretions). Trypsin-accessible 35S-labeled macromolecules appeared on the cell surface within 5-10 min, increased linearly, and plateaued by 40 min; the median transit time for 35S-labeled macromolecules to reach the cell surface was 21 min. 35S-labeled macromolecules in basal secretions increased with a similar time-course, reaching a plateau by 40 min. Incorporation of [3H]serine into the protein moiety of trypsin-accessible macromolecules occurred more slowly; trypsin-accessible 3H-labeled macromolecules were barely detectable at 1 h and increased to a maximum after 2 h, suggesting the presence of a preformed pool of nonsulfated core protein. Pretreatment with cycloheximide, an inhibitor of protein synthesis, decreased trypsin-accessible 35S-labeled macromolecules log-linearly depending on the duration of pretreatment providing an estimate of the rate of depletion of the core protein pool (t1/2 = 32 min). During continuous exposure to 35SO4, 35S-labeled macromolecules accumulated on the cell surface (trypsin-accessible compartment) for 16 h, at which point the cell-surface pool was saturated (t1/2 = 7.5 h). After pulse-labeling the cells with 35SO4 for 15 min, the 35S-labeled macromolecules disappeared continuously from the cell surface (t1/2 = 4.6 h), and 79% of the radioactivity was recovered in the medium as nondialyzable macromolecules. Release of the 35S-labeled macromolecules from the cell surface was abolished at 4 degrees C, indicative of an energy-dependent process, but multiple proteinase inhibitors did not affect the release. We conclude that sulfate is metabolized rapidly into epithelial cell-surface macromolecules, which accumulate continuously into a relatively large cell-surface pool, before they are released by an undefined energy-dependent mechanism.