The role of clathrin in the sorting of proinsulin to secretory granules,the formation of immature granules and their subsequent maturation is not known. To this end, primary rat pancreatic β-cells were infected with a recombinant adenovirus co-expressing the Hub fragment, a dominant-negative peptide of the clathrin heavy chain and enhanced green fluorescent protein(EGFP as a marker of infected cells). A population of cells expressing the highest levels of EGFP (and thus Hub) was obtained using a fluorescence-activated cell sorter (FACS). Control cells were infected with an adenovirus expressing EGFP alone. By immunofluorescence, control cells showed intense staining for both clathrin light chain and proinsulin in a perinuclear region. In cells expressing high levels of Hub, the clathrin light-chain signal was faint and diffuse in keeping with its displacement from membranes. There was, however, no detectable effect of Hub expression on proinsulin staining or disposition within the cell. Proinsulin sorting and conversion,and the fate (release and/or degradation) of insulin and C-peptide, was studied by pulse-chase and quantitative reverse phase HPLC. In both Hub-expressing and control cells, >99% of all newly synthesized proinsulin was sorted to the regulated pathway and there was no effect of Hub on proinsulin conversion to insulin. In presence of Hub there was, however, a significant increase in the percentage of C-peptide truncated to des-(27-31)-C-peptide at early times of chase as well as more extensive degradation of C-peptide thereafter. It is concluded that clathrin is not implicated in the sorting or processing of proinsulin or in regulated exocytosis of secretory granules. These results confirm a role for clathrin in the removal of proteases from maturing granules, thus explaining the increased truncation and degradation of C-peptide in cells expressing Hub.