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SUMMARY: Acinetobacter calcoaceticus, a soil isolate capable of utilizing ethanol as a sole carbon source, was cultivated in a chemostat under ethanol-limiting conditions. The pulse addition of ethanol (to less than 0.1% final concentration) to steady state cultures inhibited growth. Growth inhibition was accompanied by conversion of ethanol to acetate, after which growth resumed on the accumulated acetate and the culture returned to the original steady state. The final concentration of ethanol added and acetate accumulated were lower than levels that inhibited growth in batch culture. The rate of acetate synthesis after the pulse addition of ethanol was sufficient to support the maximum specific growth rate. (μmax) although the growth rate immediately before ethanol addition was only 47% of μmax. In batch culture acetate- or ethanol-grown cells preferentially consumed the acetate component of an ethanol-acetate mixture, but in chemostat cultures the presence of acetate did not prevent ethanol oxidation.