The primary structure of the rat insulin-degrading enzyme (IDE) was determined by cDNA analysis. Rat IDE, as well as the previously characterized homologs from human and Drosophila, contain the carboxyl-terminal consensus sequence A/S-K-L, for peroxisome targeting. A stretch of 43 bp surrounding an alternatively used polyadenylation site is highly conserved between rat and human, suggesting that it may contain important regulatory information. Northern blot analysis revealed two IDE transcripts of 3.7 and 5.5 kb in various tissues. Testis was found to be exceptional in having three different RNAs (3.7, 4.1 and 6.1 kb) at a relatively high abundance. The expression of the IDE gene in testis is correlated with sexual maturation.