Knowledge of functional soil microbial communities in agricultural systems is limited. This study assessed the effects of a 30 year-old field trial with contrasting tillage and crop rotations on soil ammonia oxidizing bacteria (AOB) and archaea (AOA), and arbuscular mycorrhizal fungi (AMF). Four different cropping systems under till and no-till were analyzed: continuous-corn (CC), soybean-soybean-corn-corn (SSCC), soybean-winter wheat-corn-corn (SWCC), and soybean-winter wheat-(with red clover cover crop)-corn-corn (SW(RC)CC). All treatments were in a corn year of their rotation in 2010 and samples were collected five times throughout the season: before (May 3) and after cultivation/planting (May 25), after UAN fertilization (June 30), before (October 12) and after (November 23) fall harvest/moldboard plowing. Surface (0-5cm) samples were collected for all treatments and an additional two sample depths (5-15cm and 15-30cm) were collected for CC and SW(RC)CC. DNA/RNA were extracted from samples within 30 days. Reverse-transcription PCR of RNA was performed immediately after extraction. Quantitative real-time PCR determined gene counts for active (RNA) and total (DNA) populations of AOB (ammonia monoxygenase gene, amoA) and AOA (crenarchaeal gene, crenamo). Throughout the study total populations of AOB averaged 24x smaller compared to AOA (P>0.001), but active AOB populations were only 40% smaller (P>0.05). Interestingly, cultivation affected active AOB and total AOA populations more than total AOB and active AOA populations. Longer-term impacts of cultivation on total AOA populations were apparent as till plots consistently exhibited lower crenamo gene copies across soil depths throughout the study (25.1% less, P=0.045). Tilled plots exhibited smaller AOB active populations only after spring (60.8% less, P=0.029) and fall (65.7% less, P=0.042) cultivation events. This suggests AOB recover quicker under cultivated soils, even if they are less abundant. Future work to quantify AMF communities and soil nutrient levels will further elucidate the effects of agronomic practices on these important soil microbes.