Mutant strains of Azotobacter vinelandii that are unable to fix nitrogen were analyzed for their ability to reduce acetylene and oxidize dithionite. The activities of Components I (Fe-Mo-protein) and II (Fe-protein), the presence of antibody cross-reacting material to each of the components and the electron paramagnetic resonance (EPR) intensities at g = 3.65 also were examined in these strains. All mutant strains so far studied that are unable to reduce nitrogen, are also incapable of reducing acetylene or oxidizing dithionite. Representatives of various nitrogenaseless mutants have been characterized. Based on activity measurements they fall into three classes: those lacking both components (I −II −), those lacking Component I (I −II +) and those lacking Component II (I +II −). Many strains have extremely low levels of activity for either component, but in some of these strains, cross-reacting material is made for one or both of the components. The EPR at g = 3.65 correlates well with the activity for Component I in several of these mutant strains, but in four of the mutants there appears to be 10-20-fold higher amounts of paramagnetic center than the nitrogen-fixing activity in in vitro tests would indicate.