Accurate determinations of stable isotope ratios require a calibration using at least two reference materials with different isotopic compositions to anchor the isotopic scale and compensate for differences in machine slope. Ideally, the [delta] values of these reference materials should bracket the isotopic range of samples with unknown [delta] values. While the practice of analyzing two isotopically distinct reference materials is common for water (VSMOW-SLAP) and carbonates (NBS 19 and L-SVEC), the lack of widely available organic reference materials with distinct isotopic composition has hindered the practice when analyzing organic materials by elemental analysis/isotope ratio mass spectrometry (EA-IRMS). At present only L-glutamic acids USGS40 and USGS41 satisfy these requirements for [delta]13C and [delta]15N, with the limitation that L-glutamic acid is not suitable for analysis by gas chromatography (GC). We describe the development and quality testing of (i) four nicotine laboratory reference materials for on-line (i.e. continuous flow) hydrogen reductive gas chromatography-isotope ratio mass-spectrometry (GC-IRMS), (ii) five nicotines for oxidative C, N gas chromatography-combustion-isotope ratio mass-spectrometry (GC-C-IRMS, or GC-IRMS), and (iii) also three acetanilide and three urea reference materials for on-line oxidative EA-IRMS for C and N. Isotopic off-line calibration against international stable isotope measurement standards at Indiana University adhered to the ‘principle of identical treatment’. The new reference materials cover the following isotopic ranges: [delta]2Hnicotine -162 to -45%, [delta]13Cnicotine -30.05 to [plus]7.72%, [delta]15Nnicotine -6.03 to [plus]33.62%; [delta]15Nacetanilide [plus]1.18 to [plus]40.57%; [delta]13Curea -34.13 to [plus]11.71%, [delta]15Nurea [plus]0.26 to [plus]40.61% (recommended [delta] values refer to calibration with NBS 19, L-SVEC, IAEA-N-1, and IAEA-N-2). Nicotines fill a gap as the first organic nitrogen stable isotope reference materials for GC-IRMS that are available with different [delta]15N values. Comparative [delta]13C and [delta]15N on-line EA-IRMS data from 14 volunteering laboratories document the usefulness and reliability of acetanilides and ureas as EA-IRMS reference materials.