Islets of Langerhans were isolated from four human kidney donors, aged 16 to 21 years, by the collagenase method described for isolation of rodent islets. So far the human islets have been kept in tissue culture, without attachment, in medium RPMI 1640 supplemented with 10% calf serum for more than 9 months, with preservation of the ability to release insulin in response to glucose stimulation. Replacement of calf serum with serum from normal human subjects did not affect B-cell survival, but resulted in elevated insulin values partly due to lower insulin degrading activity. Thus the described technique presents a valuable tool for studying chronic effects of metabolites and hormones on islet function, as well as for islet storage prior to transplantation into humans.