A mini-human insulin gene and four derivatives mutated at several regions potentially involved in the regulation of gene expression were used to generate transgenic mouse lines. The effect of these mutations on the efficiency of gene expression and cell specificity was studied using three approaches: (1) Northern blot analysis using total RNA from pancreas and other organs, (2) radioimmunoassay to detect the human C-peptide in urine samples, and (3) immunocytochemistry of pancreas sections to examine whether expression of the transgene was still specifically expressed in β-cells. Mutation of the cis-acting elements located between −238 and −206 (GCII and CTII motifs) resulted in a strong decrease of gene expression in the pancreas of transgenic mice, but it did not lead to complete extinction of the transgene expression. This region alone (−255/−202), when linked to the minimal Herpes simplex virus thymidine kinase gene (tk) promoter, failed to activate chloramphenicol acetyltransferase (CAT) gene expression in transfected insulinoma cells, while it was activated by the equivalent region of the rat insulin I gene. On the contrary, mutation of the DNA motifs located between −109 and −75 (GCI and CTI) or between −323 and −297 (CTIII) did not significantly affect the level of the human insulin gene expression in transgenic mice. Replacement of the insulin promoter (−58/+1) by the tk promoter did not alter its level of expression in transgenic mice. In all instances, expression of the different transgenes remained localized in the islet β-cells. Altogether, these results indicate that the GCII-CTII motif is an important regulatory element for efficient expression of the human insulin gene in vivo, although it alone does not allow gene expression as it would require the association of other elements.