Intact mitochondria isolated from Nicotiana tabacum cv. Bright Yellow 2 (TBY-2) cells can take up riboflavin via carrier-mediated systems that operate at different concentration ranges and have different uptake efficiencies. Once inside mitochondria, riboflavin is converted into catalytically active cofactors, FMN and FAD, due to the existence of a mitochondrial riboflavin kinase (EC 2.7.1.26) and an FAD synthetase (EC 2.7.7.2). Newly synthesized FAD can be exported from intact mitochondria via a putative FAD exporter. The dependence of FMN synthesis rate on riboflavin concentration shows saturation kinetics with a sigmoidal shape (S(0.5), V(max) and Hill coefficient values 0.32+/-0.12 microm, 1.4 nmol x min(-1) x mg(-1) protein and 3.1, respectively). The FAD-forming enzymes are both activated by MgCl(2), and reside in two distinct monofunctional enzymes, which can be physically separated in mitochondrial soluble and membrane-enriched fractions, respectively.