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Pichia stipitis NRRL Y-7124 is one of the natural yeasts best able to utilize biomass because it is able to ferment hexoses and the pentose, xylose, to economically recoverable concentrations of ethanol. To test the impact of culture conditions on inhibitor tolerance, inhibitors were spiked to growing or stationary-phase P. stipitis supplied either glucose or xylose and varying nitrogen and mineral compositions; then the ensuing specific death rate response was measured. Resistance of glucose- or xylose-grown cells to inhibitors was generally greater in stationary-phase cells than log-phase cells, despite a greater exposure of stationary cells to ethanol. Consistent with this, the specific productivity of detoxification products, furan methanol or furan-2,5-dimethanol, from respective spikes of furfural or HMF increased as cultures progressed into stationary phase. However, when xylose was the substrate, ethanol resistance behaved uniquely and was greater for log- than stationary-phase cells. Amino acid enrichment of the growth medium significantly enhanced ethanol tolerance if xylose was the carbon source, but had no impact if glucose supplied carbon. Regardless of the carbon source, amino acid enrichment of the culture medium enhanced the ability of cells to resist furfural and HMF exposure. Mineral compositions tested had little impact on inhibitor resistance except stationary-phase xylose-grown cells were more susceptible to inhibitor exposure when magnesium sulfate was excessive. Observed tolerance optimization based on specific death rate as a function of culture physiological state, carbon source, nitrogen source and mineral composition provides new knowledge supporting process designs to convert biomass to ethanol using P. stipitis. Biotechnol. Bioeng. 2009; 102: 778–790. © 2008 Wiley Periodicals, Inc.