Flow injection determinations of acetate were carried out using immobilized acetate kinase, pyruvate kinase and lactic dehydrogenase with an amperometric method. Two acetate kinases fromE. coliandB. stearothermophiluswere tested. It was found that the immobilized acetate kinase fromB. stearothermophiluswas more stable than that fromE. coli., but it is much more expensive and less available. Acetate kinase coupling at pH 7.4 using CPG aminopropyl and glutaraldehyde seems to be superior to other immobilization methods. A high immobilization yield can be obtained by immobilization of the three enzymes separately giving high conversions of all the three. Plots of current versus concentration show a useful operating range from 0.3 to 2 mM acetate with a linear response. The detection limit was 0.2 mM at a flow rate of 0.3 ml/min with 200 μl injections. The method is therefore well suited for monitoring of the level of acetate in fermentations with acetate as the carbon source.