To investigate biochemical aspects of resistance conferred by the Lr35 gene for adult‐plant resistance in wheat (Triticum aestivum L.) to leaf rust, pathogen development was related to intercellular protein composition and β‐1,3‐glucanase (EC 3.2.1.39) activities at three growth stages in infected and uninfected resistant (RL6082 [Thatcher/Lr35]) and susceptible (Thatcher) plants. Leaf rust symptoms produced by pathotype UVPrt9 of Puccinia recondita f. sp. tritici showed that resistance conferred by Lr35 was most effective at the flag leaf stage. Furthermore, fluorescence microscopy indicated that resistance was strongly associated with hypersensitive cell death of invaded tissue. According to polypeptide profiles, intercellular proteins with molecular masses of 35, 33, 31 and 26 kDa were constitutively present at higher levels in resistant than in susceptible plants at the flag leaf stage. Four intercellular proteins (35, 33, 32 and 31 kDa) serologically related to β‐1,3‐glucanase were present in resistant and susceptible genotypes during all stages of plant growth. Resistance was associated with high constitutive levels of β‐1,3‐glucanase activity. Susceptibility on the other hand was associated with low constitutive levels of β‐1,3‐glucanase, while high levels were induced by infection during more advanced stages of colonization. Our results suggest that β‐1,3‐glucanase is involved in the defense response controlled by the Lr35 gene.