These studies were undertaken to characterize the high molecular weight immunoreactive insulins in plasma and tumor extracts of two patients with insulinoma, and to determine whether they are biosynthetic precursors of proinsulin. Plasma was chromatographed on a Sephadex G-50 column and fractions assayed for IRI. Three peaks were observed, one in the void volume region (6%), a peak in the proinsulin region (25%), and the largest peak comigrating with insulin standard (69%). Insulinoma slices were incubated for 4 h and high molecular weight IRI was observed in chromatographed extracts of the tumor, as well as in extracts of the incubation medium, which comprised up to 3% of the total IRI. Gel filtration chromatography of a pre-operative fasting plasma from the second patient revealed a heterogeneous distribution of IRI, with approximately 53% in the high molecular weight region. At surgery an insulinoma was removed. Slices were incubated for 4 h with 14C-amino acids and extracts chromatogrphed. 14C-labeled protein was observed in the V0, and in the proinsulin and insulin regions. These 14C-labeled proteins were precipitated with anti-insulin serum, and it was determined that whereas 60-90% of 14C-protein in the proinsulin-insulin region was precipitated, virtually none of the 14C-protein in the V0 was immunoprecipitated with anti-insulin serum. The specific activities (CPM precipitated by anti-insulin serum/muU IRI) were 1, 11.4, and 3.1 for V0, proinsulin, and insulin regions, respectively. These results suggest that proinsulin is synthesized first, followed by insulin, and then the high molecular weight IRI. The high molecular weight IRI is not a biosynthetic precursor of proinsulin. Rechromatography of the high-molecular weight IRI in the presence of 8M urea dissociated 90% into smaller immunoreactive components which chromatographed in the proinsulin and insulin region. It was concluded that high molecular weight IRI's are present in plasma, tumor extracts, and incubation medium from insulinoma patients. These high molecular weight IRI's are not biosynthetic precursors, but either aggregates of proinsulin and insulin, or insulin bound to larger proteins. These studies do not rule out the existence in humans of a smaller rapidly-turning over precursor to proinsulin similar to the pre-proinsulin discovered in a rat insulinoma by Chan et al.