Insulin transcription factor MafA is unique in being exclusively expressed at the secondary and principal phase of insulin-expressing cell production during pancreas organogenesis, and is the only transcriptional activator present exclusively in islet cells. Here we show that ectopic expression of MafA is sufficient to induce a small amount of endogenous insulin expression in a variety of non- cell lines. Insulin mRNA and protein expression was induced to a much higher level when MafA was provided with two other key insulin activators, PDX-1 and BETA2. Potentiation by PDX-1 and BETA2 was entirely dependent upon MafA, and MafA binding to the insulin enhancer region was increased by PDX-1 and BETA2. Treatment with activin A and HGF induced even larger amounts of insulin in AR42J pancreatic acinar cells, compared with other non- endodermal cells. The combination of PDX-1, BETA2, and MafA also induced the expression of other important regulators of islet cell activity. These results support a critical role of MafA in islet cell function.