Luciferase reporter genes are widely used for the functional characterization of regulatory elements in 3'-untranslated region (3'-UTR). Using a transient expression assay system with pancreatic cell lines, we demonstrated that luciferase reporter gene constructs show not only the elements with special sequences in 3'-UTR that can affect luciferase activity, but also elements containing random sequences that were ligated into the same site. The extent of the decrease in luciferase activity was dependent on the length of the DNA fragments. Our findings strongly suggested a need to re-examine the 3'-UTR characterizations of many eukaryotic genes which have been studied to date with luciferase reporter genes.