In temperate and boreal forest ecosystems, nitrogen (N) limitation of tree metabolism is alleviated by ectomycorrhizal (ECM) fungi. As forest soils age, the primary source of N in soil switches from inorganic (NH 4 ؉ and NO 3 ؊ ) to organic (mostly proteins). It has been hypothesized that ECM fungi adapt to the most common N source in their environment, which implies that fungi growing in older forests would have greater protein degradation abilities. Moreover, recent results for a model ECM fungal species suggest that organic N uptake requires a glucose supply. To test the generality of these hypotheses, we screened 55 strains of 13 Suillus species with different ecological preferences for their in vitro protein degradation abilities. Suillus species preferentially occurring in mature forests, where soil contains more organic matter, had significantly higher protease activity than those from young forests with low-organic-matter soils or species indifferent to forest age. Within species, the protease activities of ecotypes from soils with high or low soil organic N content did not differ significantly, suggesting resource partitioning between mineral and organic soil layers. The secreted protease mixtures were strongly dominated by aspartic peptidases. Glucose addition had variable effects on secreted protease activity; in some species, it triggered activity, but in others, activity was repressed at high concentrations. Collectively, our results indicate that protease activity, a key ectomycorrhizal functional trait, is positively related to environmental N source availability but is also influenced by additional factors, such as carbon availability. In temperate and boreal forests, nitrogen (N) is the element that usually limits tree nutrition (1). To acquire sufficient N, trees form symbioses with microorganisms, including ectomycorrhizal (ECM) fungi (2), as well as shoot-endophytic bacteria (3). In soils, ECM fungi can take up N from both mineral and organic sources. Mineral N can be found as NH 4 ϩ or NO 3 Ϫ (4), while organic N can be present as part of several different organic oligomers or polymers: peptides, chitin, nucleic acids, and heterocyclic N compounds (3). Peptides are considered the dominant organic N source in forest soils (representing as much as 80% of organic N [5]), with ECM fungi typically retrieving N from this source through the use of proteases (3). Despite generally broad enzymatic capacities (6), not all ECM fungi have the ability to access peptide N, which has resulted in the classification of ECM fungi into "protein" and "nonprotein" species (7). Multiple authors have suggested that natural selection should favor traits allowing mycorrhizal fungi to utilize the most abundantly available N source in their environment (8). This would suggest that protein ECM fungal species have their ecological niche in organic-N-rich soils. Empirical support for this hypothesis has been shown by Lilleskov et al. (9), who found that ECM fungal species growing in a soil rich in mineral N had a lower a...