A high-resolution (1.16 A) X-ray structure of the nitrogenase molybdenum-iron (MoFe) protein revealed electron density from a single N, O, or C atom (denoted X) inside the central iron prismane ([6Fe]) of the [MoFe7S9: homocitrate] FeMo-cofactor (FeMo-co). We here extend earlier efforts to determine the identity of X through detailed tests of whether X = N or C by interlocking and mutually supportive 9 GHz electron spin echo envelope modulation (ESEEM) and 85 GHz electron-nuclear double resonance (ENDOR) measurements on N-14/15 and C-12/13 isotopomers of FeMo-co in three environments: (i) incorporated into the native MoFe protein environment; (ii) extracted into N-methyl formamide solution; and (iii) incorporated into the NifX protein, which acts as a chaperone during FeMo-co biosynthesis. These measurements provide powerful evidence that X; N/C, unless X in effect is magnetically decoupled from the S = 3/2 electron spin system of resting FeMo-co. They reveal no signals from FeMo-co in any of the three environments that can be assigned to X from either N-14/15 or C-13: If X were either element, its maximum observed hyperfine coupling at all fields of measurement is estimated to be A((14/15)Nx) < 0.07/0.1 MHz, A((13)Cx) < 0.1 MHz, corresponding to intrinsic couplings of about half these values. In parallel, we have explicitly calculated the hyperfine tensors for X = N-14/15/C-13/O-17, nuclear quadrupole coupling constant e(2)qQ for X = N-14, and hyperfine constants for the Fe sites of S = 3/2 FeMo-co using density functional theory (DFT) in conjuction with the broken-symmetry (BS) approach for spin coupling. If X = C/N, then the decoupling required by experiment strongly supports the "BS7' spin coupling of the FeMo-co iron sites, in which a small X hyperfine coupling is the result of a precise balance of spin density contributions from three spin-up and three spin-down (3 up arrow:3 down arrow) iron atoms of the [6Fe] prismane "waist" of FeMo-co; this would rule out the "BS6" assignment (4 up arrow:2 down arrow for [6Fe]) suggested in earlier calculations. However, even with the BS7 scheme, the hyperfine couplings that would be observed for X near g(2) are sufficiently large that they should have been detected: we suggest that the experimental results are compatible with X = N only if a(iso)(N-14/15(x)) < 0.03-0.07/0.05-0.1 MHz and ai(so)(C-13(X)) < 0.05-0.1 MHz, compared with calculated values of aiso(N-14/15(x)) = 0.3/0.4 MHz and a(iso)(C-13(X)) = 1 MHz. However, the DFT uncertainties are large enough that the very small hyperfine couplings required by experiment do not necessarily rule out X = N/C.