Low-intensity, dormant season fires were frequent and widespread in oak-hickory ( Quercus-Carya) forests of eastern North America until widespread fire suppression began in the mid-1900s. To assess how reintroduction of fire into such ecosystems might affect the activity of arbuscular mycorrhizal (AM) fungi and, thereby, predict the long-term responses of plants and soils to fire, we analyzed the content of the immunoreactive fractions of the AM-fungus-specific glycoprotein glomalin in soils taken in 1994 and 2000 from three forested watersheds in southern Ohio, USA. One watershed remained unburned, one was burned annually from 1996-1999 and one was burned twice, in 1996 and 1999. In addition, to account for the strong landscape-scale gradients of microclimate and soil that typify these watersheds, we stratified each watershed-scale treatment area into three microclimatic zones (=landscape positions) using a GIS-based integrated moisture index (IMI). In the unburned control, the concentrations of immunoreactive, easily-extractable glomalin (IREEG) and immunoreactive total glomalin (IRTG) did not change significantly over the 6-year interval between sampling times, either overall or within any of the three IMI classes. IRTG content was greatest in the mesic landscape positions and lowest in the relatively xeric landscape positions, but IREEG did not vary among landscape positions. Neither IREEG nor IRTG contents were affected by fire, nor were there significant interactions between fire and landscape position in glomalin content. Both correlation and regression analyses demonstrated significant linkages between soil glomalin content, the density/diversity of herbaceous plants, and soil N availability. Despite significant effects of fires on soil N availability and root growth, we resolved no effect of fire on AM fungal activity at this spatial scale.