Prohormones are directed from the trans-Golgi network to secretory granules of the regulated secretory pathway. It has further been proposed that prohormone conversion by endoproteolysis may be necessary for subsequent retention of peptides in granules and to prevent their release by the so-called "constitutive-like" pathway. To address this directly, mutant human proinsulin (Arg/Gly(32):Lys/Thr(64)), which cannot be cleaved by conversion endoproteases, was expressed in primary rat islet cells by recombinant adenovirus. The handling of the mutant proinsulin was compared with that of wild-type human proinsulin. Infected islet cells were pulse labeled and both basal and stimulated secretion of radiolabeled products followed during a chase. Labeled products were quantified by high-performance liquid chromatography. As expected, the mutant proinsulin was not converted at any time. Basal (constitutive and constitutive-like) secretion was higher for the mutant proinsulin than for wild-type proinsulin/insulin, but amounted to <1% even during a prolonged (6-h) period of basal chase. There was no difference in stimulated (regulated) secretion of mutant and wild-type proinsulin/insulin at any time. Thus, in primary islet cells, unprocessed (mutant) proinsulin is sorted to the regulated pathway and then retained in secretory granules as efficiently as fully processed insulin.