The availability of highly purified human islets has increased with the progressive improvement of isolation methods. This has provided opportunities to perform various in vitro studies on human islets. However, when islets are maintained in culture, the overgrowth of fibroblasts results in a reduced islet purity and often has an adverse effect on islet function. To reduce fibroblast growth and to maintain normal islet function, we have investigated a new three-dimensional culture technique using a noncoated transparent Biopore membrane insert (Millicell CM, Millipore). Islets were isolated from seven human pancreata and cultured for 2 months using this membrane insert. At various time intervals, the functional viability of is lets was assessed by measurements of insulin released into the culture medium, static incubation assays of basal and stimulated insulin release, islet insulin contents, and insulin biosynthesis. Results were compared to those of islets cultured in hydrophobic plastic petri dishes, our standard procedure. We found that the non-coated membrane does not allow islet attachment to the surface and prevents fibroblast growth, so that islets maintain a three-dimensional structure and remain in a free-floating form. Islets cultured in a membrane insert showed a function similar to or better than that of islets cultured in plastic petri dishes.