DNA insertions in the 5'-flanking region of the insulin gene have been studied by Southern-blot-hybridization techniques using a cloned gene probe in 159 unrelated Caucasians. Subjects homozygous for a large DNA insertion at this locus were found to have a higher risk of concurrence of non-insulin-dependent diabetes (NIDD) than controls (P less than 0.01). Analysis of a single large NIDD pedigree (n = 67 in four generations; 11 diabetic subjects) showed no linkage between the size of DNA inserts and diabetes (Lod score = -5.7), suggesting a separate diabetogenic locus (or loci) in this family.