Endosulfine alpha (ENSA) is an endogenous ligand of the sulfonylurea receptor 1 (SUR1) that can stimulate insulin secretion by pancreatic beta cells. Originally, an intronless gene coding for this protein was assigned to Chr. 14q, but more recent information available in public databases indicated the position of ENSA on 1q21. We show here that the 1q21 locus represents the expressed gene consisting of 6 exons, whereas the locus on Chr. 14q is apparently a pseudogene. The ENSA gene on 1q21 produces several alternatively spliced transcripts, and is located within a region linked with T2DM in diverse populations including the Pima Indians. We analyzed ENSA in this Native American population and identified seven variants, which fall into three linkage disequilibrium groups. Analysis of representative markers in over 1200 Pima Indians did not reveal any significant association with T2DM, or with differences in insulin action and insulin secretion in a subset of approximately 270 non-diabetic subjects. In addition, we did not detect any significant correlation of skeletal muscle ENSA transcript levels with differences in insulin action in 49 non-diabetic subjects. We conclude that sequence alterations in ENSA are an unlikely cause for the linkage of T2DM with 1q21-q23 in the Pima Indians.