Surface soils from long-term tillage comparison experiments at six U.S. locations were characterized for aerobic and anaerobic microbial populations and denitrification potential using an in situ acetylene blockage technique. Measurements of soil water content, bulk density, and relative differences in pH, NO-3-N, water-soluble C, and total C and N contents between tillage treatments were also determined at the time of sampling. Numbers of aerobic and anaerobic microorganisms in surface (0-75 mm) no-till soils averaged 1.35 to 1.41 and 1.27 to 1.31 times greater, respectively, than in surface-plowed soils. Bulk density, volumetric water content, water-filled pore space, and water-soluble C and organic C and N values were similarly greater for surface no-till soils compared to conventionally tilled soils. Deeper in the soil (75-300 mm), however, aerobic microbial populations were significantly greater in conventionally tilled soils. In contrast, below 150 mm, the numbers of anaerobic microorganisms differed little between tillage treatments. In no-till soils, however, these organisms were found to comprise a greater proportion of the total bacterial population than in conventionally tilled soils. Measurements of the denitrification potential from soils at three locations generally followed the observed differences in anaerobic microbial populations. Denitrifying activity, after irrigation with 15 mm of water, was substantially greater in surface 0- to 75-mm no-till soils than in conventionally tilled soils at all locations. At the 75- to 150-mm soil depth, however, the denitrification potential in conventionally tilled soils was the same or higher than that of no-till soils. In surface no-till soils, increased numbers of anaerobic microorganisms and a substantially greater denitrification potential, following irrigation, indicate the presence of less-aerobic conditions in comparison to conventionally tilled soils. This condition appears to result from greater soil bulk densities and/or water contents of no-till soils, which act to increase water-filled porosity and the potential for water to act as a barrier to the diffusion of oxygen through the soil profile.