Hydrogen bonding plays a major role in the tight binding of the FMN cofactor in flavodoxins. The present NMR investigation provides direct experimental evidence for hydrogen bonds involving the phosphate moiety of FMN in Desulfovibrio vulgaris flavodoxin. Several trans-hydrogen bond J couplings between the phosphorus nucleus and backbone amide as well as side chain hydroxyl protons of the apoprotein have been detected. It is shown that relaxation interference between H-1 chemical shift anisotropy and H-1-P-31 dipolar interactions can also lead to correlations of these nuclei in HMBC spectra. The size of the (2h)J(PH) coupling constants was determined using a simple P-31-detected quantitative J correlation experiment. For at least one amide group a scalar three-bond coupling between the phosphorus and nitrogen has been observed in a [N-15,H-1]-TROSY-type N-15-{P-31}) spin-echo difference experiment. With approximately 1.7 Hz its magnitude is larger than that of the P-31-H-1 couplings, which ranged from 0.5 to 1.6 Hz.