Toluene 4-monooxygenase (T4MO) catalyzes the NADH- and O₂-dependent hydroxylation of toluene to form p-cresol in Pseudomonas mendocina KR1. T4MO is a member of a growing family of enzymes that utilize a diiron cofactor to catalyze high-energy O₂-dependent oxidation reactions. This family includes soluble and integral membrane enzymes catalyzing desaturation reactions and hydroxylation reactions, as well as ribonucleotide reductase. Soluble diiron hydroxylases include, among others, T4MO and the soluble methane monooxygenases. While most of the diiron hydroxylases studied use three protein components, T4MO comprises four components: a 36 kDa NADH-oxidoreductase component (T4moF), a 12 kDa Rieske-type ferredoxin component (T4moC), an 11 kDa cofactorless catalytic effector protein (T4moD) and a 212 kDa diiron cofactor-containing hydroxylase component (T4moH). The presence of the Rieske-type ferredoxin, an additional electron transfer component present in T4MO that is absent in many other soluble diiron hydroxylases, introduces questions concerning the nature of complex formation and electron transfer among four-component diiron hydroxylases. Here, interactions among the four protein components of T4MO are probed using fluorescence anisotropy of a fluorophore-labeled form of T4moD (f-T4moD) engineered to include an N-terminal motif that is selective for covalent modification by the fluorescein-derived FlAsH fluorophore. High affinity complexes are detected between T4moD and T4moH, and between T4moD and T4moC. While no evidence is found for an interaction between T4moD and T4moF, excess T4moF is shown to inhibit the T4moD:T4moC complex. Moreover, a T4moD dimerization event is detected. Titration of f-T4moD with a variety of Rieske ferredoxins indicates that effector protein:ferredoxin interactions are highly specific. Biological specialization of effector protein interactions and implications for complex formation in T4MO are discussed. Funded by NSF MCB-0316232 to B.G.F.