HIV-1 drug resistance is a major clinical problem. Resistance is evaluated using in vitro assays measuring the fold change in IC₅₀ caused by resistance mutations. Antiretroviral drugs are used at concentrations above IC₅₀, however, and inhibition at clinical concentrations can only be predicted from IC₅₀ if the shape of the dose–response curve is also known. Curve shape is influenced by cooperative interactions and is described mathematically by the slope parameter or Hill coefficient (m). Implicit in current analysis of resistance is the assumption that mutations shift dose–response curves to the right without affecting the slope. We show here that m is altered by resistance mutations. For reverse transcriptase and fusion inhibitors, single resistance mutations affect both slope and IC₅₀. For protease inhibitors, single mutations primarily affect slope. For integrase inhibitors, only IC₅₀ is affected. Thus, there are fundamental pharmacodynamic differences in resistance to different drug classes. Instantaneous inhibitory potential (IIP), the log inhibition of single-round infectivity at clinical concentrations, takes into account both slope and IC₅₀, and thus provides a direct measure of the reduction in susceptibility produced by mutations and the residual activity of drugs against resistant viruses. The standard measure, fold change in IC₅₀, does not correlate well with changes in IIP when mutations alter slope. These results challenge a fundamental assumption underlying current analysis of HIV-1 drug resistance and suggest that a more complete understanding of how resistance mutations reduce antiviral activity requires consideration of a previously ignored parameter, the dose–response curve slope.