This article reports a method for the isolation of viable pancreatic islets from the human pancreas. Isolated islets were obtained from human pancreata of cadavers, patients undergoing surgical operations, and fetuses, using a freehand microdissection procedure. Viability was assessed by light microscopy of sections stained with aldehyde fuchsin and by measuring the insulin output of islets in response to a glucose stimulus in vitro using a perifusion system. Ten pieces of cadaver pancreas were studied. Islets were isolated from 6 specimens and in 5 of these were shown to respond to a glucose stimulus in vitro. Histologically the islets showed minimal damage with slight degranulation of the beta cells. Five pieces of pancreas removed at operation were studied as well. Islets were isolated in all cases, but only 2 showed a response to a glucose stimulus. Pancreata from a 26-week and 34-week human fetus were also studied. It was not possible to microdissect islets from either case, but small pieces of pancreas from the 26-week fetus were shown to respond to a glucose stimulus by producing a significant increase in insulin output.