Predisposition to type 1 diabetes and juvenile obesity is influenced by the susceptibility locus IDDM2 that includes the insulin gene (INS). Although the risk conferred by IDDM2 has been attributed to a minisatellite upstream of INS, intragenic variants have not been ruled out. We examined whether INS polymorphisms affect pre-mRNA splicing and proinsulin secretion using minigene reporter assays. We show that IVS1-6A/T (-23HphI+/-) is a key INS variant that influences alternative splicing of intron 1 through differential recognition of its 3' splice site. The A allele resulted in an increased production of mature transcripts with a long 5' leader in several cell lines, and the extended mRNAs generated more proinsulin in culture supernatants than natural transcripts. The longer mRNAs were significantly overrepresented among beta-cell-expressed sequenced tags containing the A allele as compared with those with T alleles. In addition, we show that a rare insertion/deletion polymorphism IVS1+5insTTGC (IVS-69), which is exclusively present in Africans, activated a downstream cryptic 5' splice site, extending the 5' leader by 30 bp. These results indicate that -23HphI and IVS-69 are the most important INS variants affecting pre-mRNA splicing and suggest that -23HphI+/- is a common functional single nucleotide polymorphism at IDDM2.