Identifying ‘functional' pools of soil organic matter and understanding their response to tillage remains elusive. We have studied the effect of tillage on the enriched labile fraction, thought to derive from microbes and having an intermediate turnover time. Four soils, each under three regimes, long-term arable use without tillage (NT), long-term arable under conventional tillage (CT), and native vegetation (NV), were separated into four aggregate size classes. Particle size fractions of macro- (250–2000 μm) and microaggregates (53–250 μm) were isolated by sonication and sieving. Subsequently, densiometric and chemical analyses were made on fine-silt-sized (2–20 μm) particles to isolate and identify the enriched labile fraction. Across soils, the amounts of C and N in the particle size fractions were highly variable and were strongly influenced by mineralogy, specifically by the contents of Fe and Al oxides. This evidence indicates that the fractionation procedure cannot be standardized across soils. In one soil, C associated with fine-silt-sized particles derived from macroaggregates was 567 g C m−2 under NV, 541 g C m−2 under NT, and 135 g C m−2 under CT, whereas C associated with fine-silt-sized particles derived from microaggregates was 552, 1018, 1302 g C m−2 in NV, NT and CT, respectively. These and other data indicate that carbon associated with fine-silt-sized particles is not significantly affected by tillage. Its location is simply shifted from macroaggregates to microaggregates with increasing tillage intensity. Natural abundance 13C analyses indicated that the enriched labile fraction was the oldest fraction isolated from both macro- and microaggregates. We conclude that the enriched labile fraction is a ‘passive' pool of soil organic matter in the soil and is not derived from microbes nor sensitive to cultivation.