Cysteine protease inhibitors are being studied as possible new antimalarial agents. To evaluate the potential for resistance to these compounds, we subjected chloroquine-resistant (W2 strain) Plasmodium falciparum to increasing concentrations of a vinyl sulfone cysteine protease inhibitor. After incubation with 1-200 nm morpholine urea-leucine-homophenylalanine-phenyl vinyl sulfone over approximately 8 months, highly resistant parasites ( approximately 100-fold increases in IC(50)) were selected. The vinyl sulfone-resistant parasites were also resistant to related peptidyl inhibitors, but had only modest ( approximately 2-fold) decreases in sensitivity to other cysteine protease inhibitors. Compared with the parental strain, resistant parasites showed no changes in multiplication rates, but elevations in cysteine protease activity, falcipain-2 and falcipain-3 copy numbers, transcription of falcipain genes, and levels of these target proteases in trophozoites. Resistant parasites grown in the absence of the vinyl sulfone for 12 weeks showed partial reversion, with increased inhibitor sensitivity and apparent decreases in copy numbers of falcipain-2 and falcipain-3. The sequences of falcipain-1, falcipain-2, and falcipain-3 were identical in sensitive and resistant parasites. The accumulation of a vinyl sulfone inhibitor was decreased approximately 9-fold in resistant parasites. In summary, parasites resistant to a cysteine protease inhibitor were selected, although the acquisition of high level resistance required extended exposure to the inhibitor and this resistance was somewhat unstable. Resistance was specific for the type of protease inhibitor used for the selection and appeared to be mediated both by alterations in inhibitor transport and by a previously unidentified mechanism in P. falciparum, the amplification of genes encoding targets of enzyme inhibitors.