A large number of reports have shown that the two-layer method (TLM), which employs oxygenated perfluorochemical (PFC) and University of Wisconsin (UW) solution, is superior to simple cold storage in UW in islet transplantation. However, two recent large-scale studies showed no beneficial effect of TLM compared with UW storage in human islet transplantation. We reevaluated the effect of TLM by following three groups: group 1: UW simple storage; group 2: TLM performed by multiorgan procurement teams (not specialists of islet isolation); and group 3: TLM performed by specialists of islet isolation (Noguchi and Matsumoto). There were no significant differences between groups 1 and 2, whereas islet yields were significantly higher in group 3 compared with either group 1 or 2. Our data suggest that exact, complete performance of TLM could improve the outcome of islet isolation and transplantation. In this review, we describe the mechanisms of the TLM, the procedure of preoxygenated TLM, and the several possibilities for the reasons of the discrepancy.