Rats and mice retain a duplicated insulin (I) gene. Because the duplicated gene shares only incomplete homology with the ancestral insulin (II) gene it may be regulated differently. In the studies presented here we measured changes in abundance of these distinct insulin mRNAs and their precursors in response to fasting and fasting plus a single dose of cyproheptadine, two experimental manipulations that cause changes in the level of total insulin mRNA in rats. Both diminished rat insulin II mRNA to a greater extent than rat insulin I mRNA. Rat insulin II mRNA comprised 41% of the total insulin mRNA in 0 h controls and decreased to 33% of the total insulin mRNA after a 10-h fast. Insulin II mRNA decreased to 26% of the total insulin mRNA 10 h after treatment with cyproheptadine. To determine whether these manipulations had effects on insulin mRNA synthesis, precursors for each of the two mRNAs were quantified. Fasting for 24 h had only small effects on insulin I mRNA precursor, but diminished rat insulin II pre-mRNA to 32% of the 0 h control values. One and a half hours after fasting plus cyproheptadine administration, pre-mRNA for rat insulin II levels had decreased to 38%, while rat insulin I pre-mRNA remained at levels present in 0 h controls. Levels of rat insulin I and II pre-mRNAs were both maximally depressed at 10 h, but rat insulin II pre-mRNA decreased to 3%, while rat insulin I pre-mRNA diminished to only 49% of controls. By 24 h, rat insulin I pre-mRNA had returned completely to control values, while rat insulin II premRNA levels remained depressed. These studies indicate that while mRNA synthesis from the two genes is regulated coordinately, insulin II mRNA synthesis responds to these regulating influences more rapidly and to a greater degree.