The molecular structure of 4-nitropyridine N-oxide, 4-NO -PyO, has been determined by gas-phase electron diffraction monitored by mass spectrometry (GED/MS) and by quantum chemical calculations (DFT and MP2). Comparison of these results with those for non-substituted pyridine N-oxide and 4-methylpyridine N-oxide CH -PyO, demonstrate strong substitution effects on structural parameters and electron density distribution. The presence of the electron-withdrawing –NO group in para-position of 4-NO -PyO results in an increase of the ipso-angle and a decrease of the semipolar bond length r(N→O) in comparison to the non-substituted PyO. The presence of the electron-donating –CH group in 4-CH -PyO leads to opposite structural changes. Electron density distribution in pyridine-N-oxide and its two substituted compounds are discussed in terms of natural bond orbitals (NBO) and quantum theory atoms in molecule (QTAIM). 2 3 2 2 3 3