Proinsulin conversion to insulin was studied using electrically permeabilized rat islets of Langerhans. Using high-performance liquid chromotography separation of radiolabelled islet proteins, we have demonstrated that conversion was dependent upon temperature, sensitive to pH and regulated by MgATP. Ammonium acetate was used to collapse the granular pH gradient, over a pH range of 3·5–7·4. Conversion was optimum at pH 4·5–5·5 and was reduced, but not abolished, at pH 7·4. Ca2+ (10 μm) and 4β-phorbol 12-myristate 13-acetate (500 nm), which are insulin secretagogues in permeabilized islets, caused no significant stimulation of conversion.