Summary The objective of this study was to quantify the proteolytic activity of a set of 10 diploid early intermediate heading cultivars of Lolium perenne under rumenlike conditions. A field experiment was conducted in Northern Germany, where the perennial ryegrass cultivars were grown during two growing seasons. Leaves of the first and second cut were sampled in the field, sterilized with 800 ml. l−1 ethanol solution and incubated for 0, 6, and 24 h under rumenlike conditions (darkness, 39°C, pH 6.5) without the presence of rumen microbes. Results revealed that the leaf protein content declined with increasing incubation time, confirming the involvement of plant-mediated proteolysis in the degradation process. Gel electrophoresis illustrated that the decrease in protein content is probably mainly caused by the loss of the large subunit of Rubisco (ribulose-1, 5-bisphosphate carboxylase/oxygenase), which was entirely degraded during the incubation time. Although differences among harvests and years were evident, genetic variation among the 10 diploid perennial grass samples concerning protein degradation rates and degradation characteristics was not detected.