Soils provide the substrate for important microbial mechanisms that moderate a variety of processes in both managed and natural terrestrial ecosystems. Australian soils are particularly 'stressed' and are considered to be highly weathered and nutrient deficient. This places increased pressure on the Australian cotton industry to sustainably increase productivity to support the fibre demands of a growing global population. We explored Vertosol subsoils ( 30 cm) under rotational cotton crops, measuring the distribution of soil microbial activity (SMA) and biomass (SMB) to 100 cm depth, as influenced by crop system and time, using respiration-based experiments. Seasonal SMA fluctuations were considered by capturing the long-term SMA between systems using stable oxygen isotope methodology. Our results indicate that subsoils contributed 47% of SMA, regardless of system, but subsoil SMB (26%) was constrained by resource availability. Long-term SMA was not significantly influenced by depth and system, presumably as a consequence of the shrink-swell nature of Vertosols facilitating organic matter movement down the profile. The impact of this organic matter on the activity of indigenous microorganisms below the top 30 cm of the profile highlights a need to consider deeper soil when unravelling the potential microbial benefits to our cropping systems. Additional keywords: agronomy, isotope techniques, soil microbiology, soil organic matter, Vertisol.