A cathepsin D (CD) inhibitor was searched using mouse embryonic fibroblasts deficient for CD. Synthetic DNA fragments specifically inhibited CD activity in a dose-dependent manner, but not the activities of other serine or cysteine proteinases. Cathepsin E activity was also inhibited by DNA fragments when hemoglobin was used as a substrate. CD inhibition by DNA fragments appeared to be electrostatic in nature and dependent on Tm values. Moreover, CD activity was partly inhibited by exogenously ingested DNA fragments, suggesting that DNA fragments with high Tm values are potent inhibitors of CD in vitro and partly in vivo.