Mice and rats express two nonidentical insulins from a pair of unlinked genes. We have applied a nuclease protection assay, which can sensitively quantify each of the mouse insulin mRNAs, to the resolution of the following questions concerning their expression. First, it has not been established whether alterations in expression of one or both of these genes cause differing total insulin biosynthetic capacity noted between several inbred mouse strains. These studies showed that the relative abundance of mRNAs encoding mouse insulins I and II was identical in four separate mouse strains. In spontaneously obese, hyperinsulinemic (db/db)C57BL/KsJ mice, both proinsulin I and proinsulin II mRNAs were increased relative to the levels in normal (+/db) C57BL/KsJ mice, but again the ratio of the two mRNAs did not differ. The ratio was nearly identical to that for the orthologous mRNAs in rats, indicating that the mechanisms which regulate insulin mRNAs in rodents are conserved in both genes in several mouse strains and between rodent species. This finding suggests that differences between mouse strains in insulin biosynthetic capacity result from differences in the glucose sensing/signalling mechanism at a point before coordinate gene transcription. Second, low levels of insulin synthesis have been suggested as an explanation for relatively high levels of insulin in several nonpancreatic tissues. We showed that the ribonuclease protection assay, sufficiently sensitive to measure 1/2000th the amount of insulin mRNA present in pancreas, was unable to detect insulin mRNA in salivary gland. This result indicates that the high levels of radioimmunoassayable insulin detected in salivary glands are not the result of insulin synthesis in situ.