Three methods for the preparation of islets from human fetal pancreas (17.4 +/- 1.2 wk gestational age) were compared. In each method, the pancreases were minced and followed by 1) no collagenase digestion, 2) 5 min of collagenase digestion, or 3) 14 min of collagenase digestion. The culture conditions prevented adherence of the fragments. Culture for 6-7 wk of minced fetal pancreas without collagenase digestion resulted in fragments that were a mixture of cells positive for insulin or glucagon, ducts, necrotic debris, and other unidentified cells with complete degeneration of the acinar cells. Culture of minced pancreas digested for 5 min with collagenase resulted in fragments that superficially appeared to be islets but did not have the size characteristics of human fetal islets and contained fibrous and duct elements not seen in islets. Culture of minced pancreas digested for 14 min with collagenase resulted in islets that were released into the medium and harvested by picking. These islets were morphologically similar to islets of the intact human fetal pancreas and isolated islets from rat neonatal pancreas. These islets and fragments were viable for at least 7-8 wk in culture.