To assess the effects of pregnancy on the relationships between plasma insulin and proinsulin, studies were performed during late gestation in women with normal carbohydrate metabolism or diabetes mellitus. Plasma was secured after overnight fast and 1, 2, and 3 hours following oral glucose (100 g). Samples were analyzed directly for total immunoreactive insulin (TIR) and for insulin and proinsulin following plasma fractionation by gel filtration. Fractionation disclosed that most of the normal gestational increase in basal and glucose-stimulated TIR can be ascribed to insulin rather than disproportionate increments in proinsulin-like components. Normal proinsulin/insulin relationships were also preserved in mild diabetics despite greater variability in their TIR response to glucose. Thus, mild carbohydrate intolerance during pregnancy is not attended by abnormalities in plasma proinsulin. In contrast basal proinsulin levels were elevated in 4 of 9 pregnant subjects with diabetes sufficiently severe to necessitate subsequent insulin therapy. Following glucose administration in the severe diabetics, the relative contribution from proinsulin to TIR was altered so that ratios of circulating proinsulin/insulin were increased at all levels of blood sugar. Postpartum tests of glucose tolerance in some of the normal and mildly diabetic subjects confirmed that pregnancy per se does not modify appreciably the relationships between plasma insulin and proinsulin although there may be some tendency for proinsulin to account for a smaller proportion of TIR.