The nature of nutrient limitation in large areas of temperate forest may be changing due to human activities. As N availability in these forests increases, other nutrients could increasingly constrain productivity and other ecosystem processes. To determine the nature of nutrient limitation (N, P, and Ca) in forest soils exhibiting differing N availability, we conducted three field studies in the Fernow Experimental Forest, West Virginia, USA. The first used a ubiquitous herbaceous species, Viola rotundifolia, to compare indices of N availability to the activity of root-associated phosphomonoesterase (PME) activity at two spatial scales. The second study used fertilized, root in-growth cores to assess the extent of N, P, and Ca limitation. Finally, we measured the root-associated PME activity of V. rotundifolia growing in experimental plots that have received various combinations of nutrient additions and harvest treatments. For entire watersheds, stream water nitrate concentrations were positively related to PME activities (R2 = 0.986). For small plots, PME activities were positively associated with soil nitrate availability (R2 = 0.425), and to a lesser extent with the leaf N concentrations (R2 = 0.291). Root growth into microsites fertilized with P was greater than growth into microsites fertilized with either N or Ca, especially in watersheds with high N availability. Experimental additions of N increased the root-associated PME activity of V. rotundifolia, supporting the causality of the relationship between N availability and PME activity. Collectively, our results indicate that, as N availability increases, P becomes increasingly limiting at the sites examined. Understanding how nutrient limitations change during N saturation should improve ecosystem models and better inform our attempts to mitigate any undesired effects.