The effect of the antibiotic sulfamethoxazole (SMX) on soil bacteria was studied using two methods (leucine incorporation and Biolog plates) of estimating pollution-induced community tolerance (PICT). SMX was added to an agricultural soil in a microcosm setup. The addition of different substrates (manure and alfalfa), and a non-amended soil, were also studied over 5 weeks. PICT measurements were validated by comparison with other measurements. Community structure was assessed using phospholipid fatty acid (PLFA) analysis and community-level physiological profiling (CLPP), and bacterial growth was estimated using leucine incorporation. Increased PICT was found at SMX concentrations of 20 and 500 mg SMX kg(-1) soil in samples containing manure and alfalfa, and at 500 mg SMX kg(-1) soil in non-amended soil (only concentration tested) using leucine incorporation. No effect was seen at 1 mg SMX kg(-1) soil. It was not necessary to add any substrate to increase the microbial activity in order to detect the effects of a bacteriostatic toxicant such as SMX when using measures based on bacterial growth. Direct inhibition of bacterial growth 2 days after SMX addition was correlated to PICT. No major changes in PICT due to SMX addition were found when using Biolog plates. However, there was a tendency towards increased PICT at the higher SMX concentrations in the manure-amended soil. Thus, different methods of detecting PICT have different sensitivities in detecting the toxic effects of SMX The effects of substrate amendment were reflected by changes in the microbial community, estimated using both PLEA and CLPP SMX was found to have a clear effect at the two highest levels of SMX in the manure- and alfalfa-amended soils, with an increase in fungal and a decrease in bacterial PLFAs. Little difference in the PLFA composition was found in the non-amended soil. CLPP was only affected at the highest SMX concentration. Although different variables showed different sensitivities to the effects of SMX, the results were consistent with an initial decrease in bacterial growth rates of sensitive species, which eventually transformed into more tolerant species, altering the community composition. (C) 2009 Elsevier Ltd. All rights reserved.