Quinoline oxidoreductase, an iron-sulfur molybdenum flavoprotein containing flavin adenine dinucleotide and molybdopterin cytosine dinucleotide, was purified from Pseudomonas putida 86 to homogeneity. The various electron-transfer centers of the purified enzyme were examined by electron paramagnetic resonance spectroscopy. Quinoline deuterated at position 2 was prepared by deuterodecarboxylation of 2-quinolinecarboxylic acid. Quinoline added to the enzyme elicited the Mo(V) "rapid" type Q signal arising from the complex of enzyme and substrate, whereas in oxidized quinoline oxidoreductase a Mo(V) "resting" signal was observed. EPR spectroscopy at helium temperatures below 70 K revealed the existence of two types of iron-sulfur centers, Fe-S I and Fe-S II. An organic free radical appeared upon reduction with sodium dithionite. Inactivation of the enzyme by cyanide led to the inactive desulfo quinoline oxidoreductase, which yielded another Mo(V) signal designated "slow" type Q upon reduction with dithionite. Desulfo quinoline oxidoreductase was partially reactivated by incubation with sulfide.