A simple, effective procedure using a single density layer of Hypaque-Ficoll mixture at 1.080 g/ml for rat pancreatic islet separation was proposed. The peak density of pancreatic islets was measured using Hypaque-Ficoll density gradients and was 1.0705 g/ml. The use of Hypaque-Ficoll for the preparation of a density gradient for the separation of pancreatic islets has provided a more physiological state and is more efficient than the use of multiple layers of gradients prepared from Ficoll alone.