Crop rotations and tillage practices can modify not only the total amount of organic matter (OM) in soils but also its composition. The objective of this study was to determine the changes in total organic C, microbial biomass C (MBC), carbohydrates and alkaline phosphatase activity induced by 4 yr of different rotation and tillage combinations on a Kamouraska clay in La Pocatière, Quebec. Two rotations (continuous barley (Hordeum vulgare L.) versus a 2-yr barley–red clover (Trifolium pratense L.) rotation) and three tillage treatments (moldboard plowing (MP), chisel plowing (CP) and no-tillage (NT)) were compared in a split-plot design. Total organic C was affected by the tillage treatments but not by the rotations. In the top soil layer (0–7.5 cm), NT and CP treatments had C contents 20% higher than the MP treatment. In the same soil layer, MBC averaged 300 mg C kg−1 in the MP treatment and up to 600 mg C kg−1 in the NT soil. Hot-water-extractable and acid-hydrolyzable carbohydrates were on average 40% greater under reduced tillage than under MP. Both carbohydrate fractions were also slightly larger in the rotation than in the soil under continuous barley. The ratios of MBC and carbohydrate C to total organic C suggested that there was a significant enrichment of the OM in labile forms as tillage intensity was reduced. Alkaline phosphatase activity was 50% higher under NT and 20% higher under CP treatments than under MP treatment and, on average, 15% larger in the rotation than in the continuous barley treatment. Overall, the management-induced differences were slightly greater in the top layer (0–7.5 cm) than in the lower layer of the Ap horizon (7.5–15 cm). All the properties measured were highly correlated with one another. They also showed significant temporal variations that were, in most cases, independent of the treatments. Four years of conservation tillage and, to a lesser extent, rotation with red clover resulted in greater OM in the top soil layer compared with the more intensive systems. This organic matter was enriched in labile forms. Key words: Soil management, soil quality, organic matter, carbohydrates, microbial biomass, phosphatase