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Although Brettanomyces bruxellensis continues to be a problem during red winemaking due to formation of off-odours and flavours, few interactions between intrinsic and extrinsic conditions that would limit spoilage have been identified. Using a commercially prepared Merlot wine, a 3 × 2 × 2 complete factorial design was implemented with total SO 2 (0, 60 or 100 mg l -1 ), ethanol (13% or 14·5% v v -1 ) and storage temperature (15° or 18°C) as variables. Populations of two strains of B. bruxellensis isolated from Washington wines (I1a and F3) were monitored for 100 days before concentrations of 4-ethylphenol, 4-ethylguaiacol and volatile acidity were measured. In wines with 13% v v -1 ethanol and stored at 15°C, addition of 100 mg l -1 total SO 2 resulted in much longer lag phases (>40 days) compared with wines without sulphites. At 14·5% v v -1 ethanol, culturability did not recover from wines with 100 mg l -1 total SO 2 regardless of the storage temperature (15° or 18°C). A few significant interactions were noted between these parameters which also affected synthesis of metabolites. Thus, SO 2 , ethanol concentration and storage temperature should be together used as means to reduce infections by B. bruxellensis., Significance and Impact of the Study: The potential for utilizing SO 2 along with the ethanol and storage temperature was studied to inhibit the spoilage yeast, Brettanomyces bruxellensis, during cellar ageing of red wines. This report is the first to identify the existence of interactions between these parameters that affect growth and/or metabolism of the yeast (i.e., synthesis of 4-ethylphenol, 4-ethylguaiacol and volatile acidity). Based on current and past findings, recommendations are presented related to the use of potential antimicrobial synergies between SO 2 , ethanol concentration and storage temperatures., (© 2017 The Society for Applied Microbiology.)