Although Antarctic dry valley soils function under some of the harshest environmental conditions on the planet, there is significant biological activity concentrated in small areas in the landscape. These productive areas serve as a source of C and N in organic matter redistributed to the surrounding biologically impoverished soils. We conducted a 3-yr replicated field experiment involving soil amendment with C and N in simple (glucose and NH4Cl) and complex (glycine and lacustrine detritus) forms to evaluate the resource limitations on soil microbial activity in an Antarctic dry valley. The respiratory response for all substrates was slow, with a significant but weak response to NH4Cl, followed by a more widespread response to all substrates after 2 yr and in laboratory incubations conducted 3 yr after substrate addition. This response suggests that the soil microbial community is N limited and, when that constraint is alleviated, the organisms are able to access a pool of stored C that they could not metabolize before. The effects of added C and N substrates on respiration rates under laboratory conditions were more rapid and significant than the response rates measured in situ. Because the spatial constraints that had probably limited access to soil resources by microorganisms in the field would have been removed in the laboratory incubation, this finding highlights the severe spatial constraints on access to resources in these soils.