Crotonobetaine reductase from Escherichia coli 044 K74 is an inducible enzyme detectable only in cells grown anaerobically in the presence of L(-)-carnitine or crotonobetaine as inducers. Enzyme activity was not detected in cells cultivated in the presence of inducer plus glucose, nitrate, gamma-butyrobetaine or oxygen, respectively. Fumarate caused an additional stimulation of growth and an increased expression of crotonobetaine reductase. The reaction product, gamma-butyrobetaine, was identified by autoradiography. Crotonobetaine reductase is localized in the cytoplasm, and has been characterized with respect to pH (pH 7.8) and temperature optimum (40-45 degrees C). The Km value for crotonobetaine was determined to be 1.1 x 10(-2M). gamma-Butyrobetaine, D(+)-carnitine and choline are inhibitors of crotonobetaine reduction. For gamma-butyrobetaine (Ki = 3 x 10(-5M)) a competitive inhibition type was determined. Various properties suggest that crotonobetaine reductase is different from other reductases of anaerobic respiration.