Because 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) is known to activate the biosynthesis of numerous proteins in various tissues, experiments were undertaken to compare the influence of 1,25(OH)2D3 in vitro on both the secretion and biosynthesis of insulin in islets of Langerhans from both 4-week vitamin D3-deficient rats and normal rats. Islets were either incubated or perifused after a 6-h induction period in the presence of various concentrations of 1, 25(OH)2D3 from 10(-12) M, which was inactive in controls, to 10(-6) M. Experiments were performed in the presence of a non-labelled amino acid mixture, to favour protein synthesis. Tritiated tyrosine was added as tracer during glucose stimulation. The newly synthesised proteins, labelled with [3H]tyrosine, were extracted by an acid-alcohol method and separated by gel chromatography adapted for low-molecular-weight proteins. Even in the presence of the amino acid mixture, the insulin response of the islets to 16.7 mM glucose was decreased by vitamin D3 deficiency and improved by 1,25(OH)2D3. This beneficial effect did not occur in basal conditions, but only during glucose stimulation, and was observed in both phases of insulin release. Moreover, these effects disappeared in the presence of 5x10(-4 )M cycloheximide, a protein biosynthesis inhibitor. Islets from vitamin D3-deficient rats exhibited a general decrease in the amount of de novo biosynthesised proteins and of [3H]tyrosine-labelled insulin and proinsulin fractions. A 6-h period of 1,25(OH)2D3 induction significantly improved the amount of de novo biosynthesised proteins, and particularly of newly synthesised insulin in response to a 2-h glucose stimulation. Calculation of the rate of conversion of newly synthesised proinsulin-like material to insulin as the [3H]insulin/[3H]proinsulin-like material ratio provided evidence for a dose-dependent increase, induced by 1, 25(OH)2D3, that could exceed that of normal islets. These data support the hypothesis that 1,25(OH)2D3 in vitro not only facilitated the biosynthetic capacity of the beta cell - which was highly induced during a 16.7-mM glucose stimulation, via a global activation of islets protein biosynthesis - but also produced an acceleration of the conversion of proinsulin to insulin.