The purpose of these experiments was to determine the relative content and biosynthetic rate of insulins I and II under various experimental conditions. The two insulins were quantitated by polyacrylamide gel electrophoresis, electrotransfer to nitrocellulose paper, photoaffinity crosslinking, and immunodetection with anti-insulin antibody and 125I-labeled protein A. The ratio (mean +/- SEM) of insulins, I/II, was 1.2 +/- 0.2 in Wistar-Furth rats fasted for 4 days, 1.6 +/- 0.2 in normal rats, and 5.5 +/- 0.8 in growth hormone-tumor-bearing hyperinsulinemic rats (P less than 0.01). The increase in content of rat insulin I compared to II in the growth hormone-tumor-bearing animals was confirmed by radioimmunoassay of gel slices. To determine whether the difference in contents of rat insulins I and II in the hyperinsulinemic rats was due to increased biosynthesis or a different turnover rate, isolated rat islets were incubated in [3H]leucine for 4 hr with 5.5 mM or 16.0 mM glucose in the incubation medium. Glucose stimulated insulin biosynthesis greater than 8-fold. The ratio of synthesis of rat insulin I relative to II was 0.9 +/- 0.1 at 5.5 mM glucose and 9.8 +/- 3.3 (P less than 0.01) at 16.0 mM glucose. Therefore, under conditions that stimulate insulin biosynthesis, there was a marked preferential synthesis of rat insulin I relative to II. These studies suggest that the two rat insulin genes are expressed independently and that, under stimulatory conditions, there is preferential expression of the rat insulin I gene.