Abstract: 2H-NMR spectra of perdeuterated octadecanoic acid (C18AC) and dotriacontane (C32AN) added to isolated and subsequently recrystallized cuticular wax from barley (Hordeum vulgare L.) leaves were recorded between 298 and 328 K. They were compared to calorimetric excess heat capacity profiles. The NMR-data revealed the presence of both an isotropic and a rigid wax component at temperatures below 313 K. At temperatures above 318 K all labels are in the fast motion regime indicating a transition in the host matrix of the labels. The presence of the surfactant C6E3 reduced the order of the C18AC-label but did not influence the order of the long chain alkane label (C32AN). Most surprisingly, calorimetry revealed that most thermotropic events take place above the apparent melting observed in NMR. Furthermore, the macroscopic softening and melting of the wax took place in the same temperature regime as in the calorimetric experiments. The excess heat capacity traces were complex and indicated a heterogeneous structural composition of the barley wax. We interpreted the apparent conflict between the NMR and the calorimetric results by assuming a crystalline host matrix, formed by C26-alcohol, the major molecular component of the wax. Within the crystal compartments there may exist an amorphous matrix with some crystalline microdomains of other wax components, including the NMR-labels. The melting of the amorphous environment leads to fast motional narrowing of the NMR spectral line of the microdomains without melting of the macroscopic structure. The measurements of diffusion coefficients (D) of radiolabelled C18AC and C32AN gave additional insight into the microstructure of the way architecture. Identical results in terms of D were obtained when radiolabelled C18AC was added to the wax from either the exterior after recrystallization or when it was recrystallized together with the wax. It is concluded that in both cases the radiolabelled molecule is located in an amorphous wax phase, which forms a percolating path through the wax and is thus also accessible to the surfactant C6E3. In contrast, D of C32AN in barley wax was about 2400 times higher when C32AN was added to recrystallized wax from the exterior compared to wax samples recrystallized together with C32AN. This indicates that in the case of C32AN the alkane is trapped within separate microdomains of the wax during recrystallization and thus it remains essentially immobile, whereas it possesses a high degree of mobility when it is added from the exterior where it has only access to the percolating amorphous wax phase.