To study the mechanism of gel-forming mucin packaging within mucin granules, we generated human mucous/goblet cells stably expressing a recombinant MUC5AC domain fused to green fluorescent protein (GFP). The fusion protein, named SHGFP-MUC5AC/CK, was accumulated within the granules together with the native MUC5AC. Inhibition of protein synthesis or the disorganization of the Golgi complex did not result in diminished SHGFP-MUC5AC/CK intragranular signals, consistent with long-term storage of the fusion protein. However, SHGFP-MUC5AC/CK was rapidly discharged from the granules upon incubation of the cells with ATP, an established mucin secretagogue. Several criteria indicated SHGFP-MUC5AC/CK was not covalently linked to endogenous MUC5AC. FRAP analysis suggested that the SHGFP-MUC5AC/CK intragranular mobile fraction (Mf) and mobility, respectively, were significantly smaller than in the endoplasmic reticulum lumen. Incubation of the cells with bafilomycin A1, a specific inhibitor of the vacuolar H+-ATPase, did not alter the fusion protein mobility, although it significantly increased (~20%) intragranular SHGFP-MUC5AC/CK Mf. In addition, the granules in bafilomycin-incubated cells typically exhibited a heterogeneous intralumenal distribution of the fluorescent fusion protein. These results are consistent with a model of the mucin granule intralumenal organization with two phases: a) a mobile phase, in which secretory proteins diffuse as in the endoplasmic reticulum lumen but at a lower rate; and b) an immobile phase or matrix in which proteins are immobilized by non-covalent, pH-dependent interactions. An intralumenal acidic pH, maintained by the vacuolar H+-ATPase, is one of the critical factors for secretory protein binding to the immobile phase and also for its organization.