AbstractMolecular size and net charge of isoforms of pathogenesis‐related (PR) chitinase, β‐1,3‐glucanase and peroxidase were studied in uninfected barley (Hordeum vulgare L., ν. Karat) leaves and in barley leaves infected with the pathogenic fungus Drechslera teres f. teres (Sacch.) Shoem. Molecular characteristics were determined by time‐dependent polyacrylamide gradient gel electrophoresis under native conditions and by applying an extended version of the computer program MOL‐MASS (Rothe, G. M., Weidmann, H., Electrophoresis 1991, 12, 703–709). Uninfected barley leaves contained predominantly one peroxidase isozyme but also three very weak peroxidases. Activities of all of these three peroxidases increased considerably after infection with Drechslera teres. The molecular masses of peroxidases 1 and 3 were estimated to be 38 ± 5 and 42 ± 7 kDa and their apparent valences at pH 8.4 were Z = 3.13 and 3.20, respectively. Amongst the chitinase isoforms, chitinase 1 and chitinase 2 appeared after infection, while chitinase 3 was also observed in uninfected leaves of barley. The molecular mass of chitinase 3 (31 ± 6 kDa; f/fo = 1.20) was larger than that of chitinase 1 (20 ± 2 kDa; f/fo = 1.04) and chitinase 2 (23 ± 3 kDa; f/fo = 1.06). The valence of constitutive chitinase 3 (Z = 1.44 ± 0.81) at pH 8.4 was lower than that of adaptive chitinase 1 (Z = 3.27 ± 1.02) and chitinase 2 (Z = 2.96 ± 1.38). Infection of barley leaves with Drechslera teres also induced the hydrolytic enzyme β‐1,3‐glucanase 1; β‐1,3‐glucanase 2 appeared in uninfected and in infected leaves. Constitutive β‐1,3‐glucanase 2 was smaller (molecular mass 19 ± kDa; f/fo = 1.05) than adaptive β‐1,3‐glucanase 1 (molecular mass 26 ± 4 kDa; f/fo = 1.07). The valence of adaptive β‐1,3‐glucanase 1 (Z = 9.58 ± 4.17) was approximately threefold that of β‐1,3‐glucanase 2 (Z = 2.80 ± 0.93).