In the developing rat pancreas from about day 15 until birth there is a marked increase in the concentration of insulin. The present experiments were designed to determine whether enhanced insulin biosynthesis is accompanied by a coordinate increment in proinsulin mRNA. Using a sensitive RNA filter hybridization technique and a cloned rat proinsulin 32P-cDNA as a hybridizing probe, the concentration of proinsulin mRNA was measured. A greater than 900-fold accumulation of proinsulin mRNA was found, which closely paralleled that of immunoreactive insulin. Proinsulin mRNA and immunoreactive insulin were highest 1-2 days after birth, more than eightfold higher than that in adult pancreas. The patterns of digestion by a number of restriction endonucleases of the two nonallelic insulin genes were identical in adult and fetal DNA and in DNA from an insulinoma. We found no evidence of major gene rearrangement during embryogenesis as a prerequisite to insulin gene expression. Glucose stimulated insulin biosynthesis at the earliest time examined (day 15 of gestation). Insulin biosynthesis was enhanced almost fourfold while proinsulin mRNA differed by less than 20% during a 2-h incubation. This suggested that the short-term effect of glucose on insulin biosynthesis in the developing rat pancreas occurs at the level of translation of existing mRNA, similar to that noted in adult pancreatic islets. These data taken together suggest that the same controls for insulin biosynthesis in the adult exist at these early times in pancreatic development and that the controls for insulin biosynthesis appear coordinately with the appearance of increased insulin during endocrine pancreatic differentiation.