The influence of arginine, lysine, tolbutamide and glibenclamide on (pro‐)insulin biosynthesis and release of newly synthesized (pro‐)insulin was studied in isolated islets of rat pancreas. Islets were incubated with ³H‐leucine and glucose in the presence and absence of the test agents. Proinsulin and insulin of islets and incubation media were separated by gel filtration on Sephadex G 50. Estimations were carried out for radioactivity and immunoreactivity for insulin. All four test substances were able to enhance insulin release whereas no stimulation of leucine incorporation into (pro‐)insulin was found. Arginine and tolbutamide even markedly reduced (pro‐)insulin synthesis. Conversion of proinsulin to insulin was not affected by any of the test agents. For studying the influence of the 4 substances on secretion of newly synthesized (pro‐)insulin two experimental models were used: 1) Labelling of the islets in the presence of the test agents, followed by uniform stimulation with glucose alone in the presence or absence of Ca⁺⁺. 2) Addition of the 4 test substances after uniform prelabelling of the islets. 1) Presence of arginine and sulfonylureas during labelling resulted in a significantly enhanced relative fractional release of newly synthesized (pro‐)insulin, although the bulk of secreted hormone appeared to stem from the storage pool also under these conditions. The enhanced fractional release was persistent also during the postlabelling period when the islets had been labelled in the presence of arginine or glibenclamide. On the other hand, a decreased release of newly synthesized (pro‐)insulin was observed during the postlabelling period in islets labelled in the presence of tolbutamide. Lysine was without significant effects in both periods. Omission of calcium ions during the postlabelling period inhibited the release of both immunoreactive and radioactive hormone. 2) When amino acids or sulphonylureas were added after prelabelling no signifcant changes were found in the specific radioactivity of released (pro‐)insulin or in the fractional release of newly synthesized hormone. Enhanced release of fresh granules from the beta cell might explain the increased fractional release of newly synthesized (pro‐) insulin when labelling is carried out in the presence of arginine and sulphonylureas, especially glibenclamide.