We examined the effect of reduced tillage on the accumulation of fungal-versus bacterial-derived organic matter within the soil matrix by quantifying the amino sugars glucosamine (Glc), galactosamine (Gal), and muramic acid (MurA) in aggregate-size fractions isolated from no-tillage (NT) and conventional-tillage (CT) soil. Intact soil cores (0- to 5- and 5- to 20-cm depth) were collected from the long-term tillage experiment at Horseshoe Bend in Athens, GA. Four water-stable aggregate-size fractions were isolated: large macroaggregates (>2000 [micro]m), small macroaggregates (250-2000 [micro]m), microaggregates (53-250 [micro]m), and the silt + clay fraction (250 [micro]m), microaggregates contained within macroaggregates, and the silt + clay fraction. Amino sugars were extracted from all fractions, purified, and analyzed by gas chromatography. Fungal-derived amino sugar C (FAS-C) comprised 63%, while bacterial-derived amino sugar C (BAS-C) accounted for 37% of the total amino sugar C pool under both tillage treatments. No-tillage soil contained 21% more amino sugar C than the CT soil across the entire plow layer. Both, the percentage of total organic C as FAS-C and BAS-C were significantly higher in the silt + clay fraction of NT versus CT soil. The percentage of total organic C as FAS-C was significantly higher in small macroaggregates of NT versus CT soil due to a preferential accumulation of FAS-C in the microaggregates contained within these macroaggregates. These results indicate that microbial-derived C is stabilized in NT soils, due primarily to a greater fungal-mediated improvement of soil structural stability and concurrent deposition of fungal-derived C in microaggregates contained within macroaggregates.