The purpose of these studies was to determine whether insulin gene expression at the level of proinsulin mRNA could be studied in human pancreas. RNA were isolated from autopsy specimens and analyzed by RNA-blot hybridization with various 32P-human insulin gene probes spanning either the entire gene or the second intervening sequence. A major band 0.62 kilo-bases (kb) in length accounted for over 95% of the mRNA, consistent in size with presumed mature proinsulin mRNA. In addition, minor bands of 1.5 and 1.3 kb were seen, consistent with an initial gene transcript containing both intervening sequences and with a processed intermediate. The 1.5- and 1.3-kb RNA were confirmed to be proinsulin mRNA precursors by hybridization specifically with the IVS II probe. Total RNA and polyadenylated RNA from five normal pancreata and two insulinomas revealed the same pattern. This method provides a means of determining whether altered insulin gene expression is one cause of diabetes.