Microbial communities exist and are active in a complex 3-D physical framework which can cause a variety of micro-environments to develop that are more or less suitable for microbial growth, activity and survival. If there is a significant microbial biogeography at the pore scale in soil, then the relationship between microbial diversity and ecosystem function is likely to be affected by micro-environmental variations at the pore scale. In this laboratory study we show that there is a significant pore-scale microbial biogeography by labelling microbial communities in different pore size classes of undisturbed soil cores with 13C-labelled fructose (a soluble, labile substrate). This was achieved by adding the substrate solution to the samples at different matric potentials (-100 kPa, -3.15 kPa and -1 kPa; placing the substrate in pores with maximum diameter of 0.97, 9.7 and 97 μm, respectively) and incubating the samples for two weeks. The mineralisation of soil organic carbon and fructose was measured as CO2 and 13C-CO2, respectively, in the jar headspace throughout the incubation. At the end of incubation we analysed the total microbial community structure using PLFA. The structure of microbial communities in different pore size classes was measured by PLFA stable isotope probing. Total PLFA profiles suggested that there was little effect of the incubation conditions on microbial community structure. However, labelled PLFA profiles showed that microbial community structure differed significantly among pore size classes, the differences being due primarily to variations in the abundance of mono-unsaturated lipids (Gram-biomarkers) and of the fungal biomarker (C18:2(9,12)). This is the first evidence for a significant microbial biogeography at the pore scale in undisturbed soil cores.