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Ross Island, Antarctica, is home to the southernmost breeding colonies of Ad,lie penguins, with associated ornithogenic nutrient enrichment to soil. Most of our knowledge about ornithogenic soils in Antarctica comes from studies of the Peninsula region, with much less known about soils in the Ross Sea region of continental Antarctica where climate is more extreme and biological communities are less diverse. We measured soil properties (pH, salinity, total and organic C and N, extractable mineral N and P), microbial biomass, and CO2 flux at three penguin rookeries on Ross Island differing in colony size and along gradients of penguin activity extending from the colony to soils lacking visible penguin influence. We determined both in situ and potential rates of CO2 flux, as well as the nutrient limitations on C cycling by fertilizing soils with N and P, in controlled laboratory incubations. Larger pools of soil C, N, and P along with elevated soil CO2 flux were found in soils with high penguin activity as compared to areas of low or no apparent activity. In laboratory incubations, CO2 flux was stimulated by addition of C with N or P, but not with N or P alone, suggesting that even with the deposition of large amounts of penguin guano, soil biota are not released from C limitation. The influence of penguin activity was not directly related to colony size but rather to the intensity of activity within the rookery (e.g., frequency of penguin presence). Our results show that penguin-influenced soils are biogeochemical hot spots where altered nutrient pools are associated with increased CO2 flux.