Hepatocyte nuclear factor 4alpha (HNF4alpha) is a critical transcription factor for pancreas and liver development and functions in islet beta cells to maintain glucose homeostasis. Mutations in the human HNF4A gene lead to maturity onset diabetes of the young (MODY1) and polymorphisms are associated with increased risk for type 2 diabetes mellitus (T2DM). Expression of six HNF4alpha variants, three each from two developmentally regulated promoters, has been firmly established. We have now detected a new set of HNF4alpha variants designated HNF4alpha10-12 expressed from distal promoter P2. These variants, generated by inclusion of previously undetected exon 1E (human=222 nt, rodent=136 nt) following exon 1D have an altered N-terminus but identical remaining reading frame. HNF4alpha10-alpha12 are expressed in pancreatic islets (and liver) and exhibit transactivation potentials similar to the corresponding alpha7-alpha9 isoforms. DNA-binding analyses implied much higher protein levels of HNF4alpha10-alpha12 in liver than expected from the RT-PCR data. Our results provide evidence for a more complex expression pattern of HNF4alpha than previously appreciated. We recommend inclusion of exon 1E and nearby DNA sequences in screening for HNF4alpha mutations and polymorphisms in genetic analyses of MODY1 and T2DM.