Some characteristics of root-secreted proteases were studied. We measured their molecular weights and mechanism of BSA digestion in comparison to endogenous root proteases. We related these studies to culture medium N composition. The seedlings of Allium porrum L. (cv. Bartek) were cultivated on MS medium, MS without inorganic nitrogen (MS-IN), and MS without IN, but with 0.1% casein (MS-IN + 0.1% casein). Electrophoretic study showed that root-secreted proteases had one isoform with a mol wt of 45 kD, regardless of medium N composition. Difference in molecular weights of root-secreted proteases and endogenous root proteases active under used conditions (>66 kD) provide us another strong evidence that root-secreted proteases were not just leaking from the roots, but they were secreted. Proteases exuded by roots degraded BSA in a similar way as endogenous proteases, with only one SDS-PAGE-detectable product of degradation. Our results may be a powerful tool in the extraction and purification of these enzymes and also in proteomic studies.