The FMN-binding domain of human NADPH-cytochrome P450 reductase, corresponding to exons 3-7, has been expressed at high level in an active form and labelled with 13C and 15N. Most of the backbone and aliphatic side-chain 1H, 15N and 13C resonances have been assigned using heteronuclear double- and triple-resonance methods, together with a semiautomatic assignment strategy. The secondary structure as estimated from the chemical shift index and NOE connectivities consists of six alpha-helices and five beta-strands. The global fold was deduced from the long-range NOEs unambiguously assigned in a 4D 13C-resolved HMQC-NOESY-HMQC spectrum. The fold is of the alternating alpha/beta type, with the five beta-strands arranged into a parallel beta-sheet. The secondary structure and global fold are very similar to those of the bacterial flavodoxins, but the FMN-binding domain has an extra short helix in place of a loop, and an extra helix at the N-terminus (leading to the membrane anchor domain in the intact P450 reductase). The experimental constraints were combined with homology modelling to obtain a structure of the FMN-binding domain satisfying the observed NOE constraints. Chemical shift comparisons showed that the effects of FMN binding and of FMN reduction are largely localised at the binding site.