P450 RhF from Rhodococcus sp. NCIMB 9784 is the first example of a new class of cytochrome P450 in which electrons are supplied by a novel, FMN and Fe/S containing, reductase partner in a fused arrangement. We have previously cloned the gene encoding the enzyme and shown it to encode an N-terminal P450 domain fused to a reductase domain that displays similarity to the phthalate family of oxygenase reductase proteins. A reductase of this type had never previously been reported to interact with a cytochrome P450. In this paper we describe the purification and partial characterization of P450 RhF. We show that the enzyme is self-sufficient in catalyzing the O-dealkylation of 7-ethoxycoumarin. The P450 RhF catalyzed O-dealkylation of 7-ethoxycoumarin is inhibited by several compounds that are known inhibitors of cytochromes P450. Pre-steady-state kinetic analysis indicates that P450 RhF shows a 500-fold preference for NAPDH over NADH in terms of Kd value (6.6 µM versus 3.7 mM respectively). Potentiometric studies show reduction potentials of -243 mV for the two-electron reduction of the FMN and -423 mV for the heme (in the absence of substrate).