Ten transgenic mouse lines harboring the -346/-103 fragment of the rat insulin I enhancer linked to a heterologous promoter and a reporter gene (Eins-Ptk-CAT construct) were produced. Expression of the hybrid transgene was essentially observed in pancreas and to a lesser extent in brain. These results indicate that the rat insulin I promoter is dispensable for pancreatic expression. This insulin gene sequence is the shortest fragment described as conferring tissue-specific expression in transgenic mice. Two short homologous sequences in the rat insulin I enhancer fragment used, IEB2 and IEB1, have been described as playing a dominant role in the regulation of HIT hamster insulinoma cell-specific transcription of the insulin gene (1). We investigated whether the combination of IEB2 and IEB1 sequences is sufficient to confer specific expression in transgenic mice to a IEB2-IEB1-Ptk-CAT gene construct. No CAT activity was observed neither in pancreas nor in any other organ examined in 19 different transgenic mice. Moreover in transient expression experiments in RIN2A rat insulinoma cells, the IEB sequences had a very weak or no enhancer activity. These observations contribute to the conclusion that DNA regulatory elements other than the IEB sequences are necessary for gene expression in vivo.