AtT20 (mouse pituitary corticotroph) cells were stably transfected with human proinsulin cDNA. Clone H12 displayed low basal release of insulin-like immunoreactivity (< 1% cell content/30 min) with 17-fold stimulation by isobutylmethylxanthine/forskolin. Clone H23, by contrast, showed higher basal release (2.8% cell content/30 min) and only 6-fold stimulation. To follow the kinetics of conversion and release of only newly synthesized proinsulin, cells were pulse-chased, and labeled proinsulin-related products were analyzed by high pressure liquid chromatography. In the cells of both clones, [3H]proinsulin was converted to insulin with des-31,32-split proinsulin as the only detectable intermediate. While basal release of labeled products from H12 cells was low (3%/60 min), it was rapid and elevated from H23 cells (12.5% by 30 min and 24.8% by 60 min of chase) with [3H]des-31,32-split proinsulin the predominant molecular form. Stimulation of [3H] insulin release increased with time, reaching 3.8-fold by 90 min of chase, whereas that of [3H]des-31,32-split proinsulin was approximately 1.5-fold regardless of the chase time. Rapid secretion of newly synthesized products that is insensitive to secretagogues is the hallmark of the constitutive pathway. Thus in H23 cells an unusually large amount of proinsulin is diverted to the constitutive pathway, where it is partially converted to des-31,32-split proinsulin before release.