The homodimeric NADH:flavin oxidoreductase from Aminobacter aminovorans is an NADH-specific flavin reductase herein designated FRDAa. FRD Aa was characterized with respect to purification yields, thermal stability, isoelectric point, molar absorption coefficient, and effects of phosphate buffer strength and pH on activity. Evidence from this work favors the classification of FRDAa as a flavin cofactor-utilizing class I flavin reductase. The isolated native FRDAa contained about 0.5 bound riboflavin-5′-phosphate (FMN) per enzyme monomer, but one bound flavin cofactor per monomer was obtainable in the presence of excess FMN or riboflavin. In addition, FRDAa holoenzyme also utilized FMN, riboflavin, or FAD as a substrate. Steady-state kinetic results of substrate titrations, dead-end inhibition by AMP and lumichrome, and product inhibition by NAD+ indicated an ordered sequential mechanism with NADH as the first binding substrate and reduced FMN as the first leaving product. This is contrary to the ping-pong mechanism shown by other class I flavin reductases. The FMN bound to the native FRDAa can be fully reduced by NADH and subsequently reoxidized by oxygen. No NADH binding was detected using 90 μM FRDAa apoenzyme and 300 μM NADH. All results favor the interpretation that the bound FMN was a cofactor rather than a substrate. It is highly unusual that a flavin reductase using a sequential mechanism would require a flavin cofactor to facilitate redox exchange between NADH and a flavin substrate. FRDAa exhibited a monomer-dimer equilibrium with a Kd of 2.7 μM. Similarities and differences between FRD Aa and certain flavin reductases are discussed.