Esters contribute a distinctive fruity aroma to beer. Their production is affected by numerous factors including yeast strain characteristics, medium composition and fermentation conditions. In the past, the main focus has been on the synthesis of these aroma compounds during beer production. Recently, it has been suggested that the esterase activity of yeast could also play a significant role in determining the final ester level of products such as membrane filtered beer and bottle re-fermented beer. The aim of this study was to determine the influence of the yeast strain, fermentation conditions and medium composition on brewing yeast esterase activity. Two in vivo esterase assays using either hydrolysis of p-nitrophenyl acetate (extracellular esterase activity) or hydrolysis of beta-naphtyl caprylate (total esterase activity) as substrates, were used to investigate the effect of medium composition and fermentation conditions on esterase activities of lager and ale yeasts. The esterase activity profiles of ale and lager brewing strains were monitored during fermentation. Both ale yeast and lager yeast showed similar behaviours. Extracellular esterase activity and total esterase activity decreased significantly after pitching with a minimum for the two activities at mid-exponential growth phase, followed by an increase. When the yeast growth entered the stationary phase, extracellular esterase activity kept increasing while the total esterase activity remained constant. Under identical culture conditions, total esterase level was found to be similar between ale and lager yeast strains. There were, however, significant differences in esterase activities between yeast strains within each group (ale or lager yeast strains). Medium FAN (free amino nitrogen) and original extract had little effect on esterase activities. Lower total esterase activity was observed at low fermentation temperature. Aerobic/anaerobic conditions had little effect on total esterase activity. Lack of oxygen favoured high level of extracellular esterase activity at the end of fermentation.