We reported that several HIV protease inhibitors (HIV-PIs) interfere with the endoproteolytic processing of two farnesylated proteins, yeast a-factor and mammalian prelamin A. We proposed that these drugs interfere with prelamin A processing by blocking ZMPSTE24, an integral membrane zinc metalloproteinase known to play a critical role in its processing. However, because all of the drug inhibition studies were performed with cultured fibroblasts or crude membrane fractions rather than on purified enzyme preparations, no definitive conclusions could be drawn. Here, we purified Ste24p, the yeast ortholog of ZMPSTE24, and showed that its enzymatic activity was blocked by three HIV-PIs (lopinavir, ritonavir, and tipranavir). A newer HIV-PI, darunavir, had little effect on Ste24p activity. None of the HIV-PIs had dramatic effects on the enzymatic activity of purified Ste14p, the prenylprotein methyltransferase. These studies strongly support our hypothesis that HIV-PIs block prelamin A processing by directly affecting the enzymatic activity of ZMPSTE24, and in this way they may contribute to lipodystrophy in individuals undergoing HIV-PI treatment.