An intracellular esterase from Lactobacillus casei subsp. casei IFPL731 was purified 1000-fold by ion exchange chromatography and gel filtration chromatography. The relative molecular mass of the native enzyme was 105 kDa, while the subunit molecular mass was estimated to be 38 kDa. The esterase hydrolysed tributyrin and had a preference for esters of short-chain fatty acids (butyrate, caproate and caprylate), while it did not hydrolyse palmitate and sterate esters. The apparent Michaelis-Menten constant of the enzyme on p-nitrophenyl butyrate was 0·3 mmol l−1 while on p-nitrophenyl caprylate, it was 0·04 mmol l−1. The esterase was active over a broad range of pH and temperature values, and retained about 50% of maximal activity at pH 5·0 and 12 °C. Activity was strongly inhibited by 5 mmol l−1 phenylmethylsulphonyl fluoride, β-mercaptoethanol and N-ethylmaleimide, and was stimulated by Zn2+ at 1 mmol l−1.