Isolated rat islets of Langerhans were pulse-labeled (5 min, [3H]leucine) and then exposed to 10 or 50 mM tris(hydroxymethyl)aminomethane (Tris) at pH 7.4 during an 85-min chase period. There was a dose-related inhibition of the conversion of labeled proinsulin to insulin by Tris. At 50 mM, Tris also inhibited the release of newly synthesized (labeled) proinsulin and insulin. These inhibitory effects of Tris were almost absent if the islets were exposed to 50 mM Tris during only the last 60 min of the 85-min chase period. Both proinsulin and total islet protein synthesis (as indexed by incorporation of [3H]leucine) were inhibited acutely by 50 mM Tris (5-min exposure); after 85 min of exposure to 50 mM Tris, the inhibition of proinsulin biosynthesis was more marked than that of total islet protein. In contrast to its effects on newly synthesized products, 50 mM Tris failed to inhibit the release of immunoreactive insulin during an 85-min incubation. However, when islets were exposed to 50 mM Tris for a longer period, a partial inhibition of immunoreactive insulin release was observed as from 120 min. Insulin released from islets consists of a mixture of older stored material and of newly synthesized products, the latter being released preferentially. These results are consistent with a selective effect of 50 mM Tris on the production of newly synthesized insulin. During the first 120 min of exposure to Tris, islet reserves of newly synthesized products will be depleted thereby leading to a new, reduced, rate of release of immunoreactive material consisting only of older insulin stores.