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The aim of the study was to assess if pedogenic processes such as mineral weathering and lessivage, other than organic matter accumulation, can affect soil microbial population and enzyme activities. This study examines six soil profiles located in a karst region of the North-Eastern Italian Alps and characterized by a vertical textural differentiation due to lessivage. For each soil, four pedological layers were recognized according to the dominant soil forming process: i) the top soil (Tp layer), formed by A and AB horizons, characterized by organic matter accumulation; ii) the subsurface eluviated layer (Elu layer), comprising AE and EB horizons; iii) the layer dominated by the in-situ mineral weathering (Wh layer), made by Bw horizons; iv) the deepest layer (Ls), subjected to clay illuviation and comprised by Bt horizons. In the upper layers (Tp and Elu), because of the low pH, weathering also occurred, as indicated by the presence of disordered smectite and by the high values of pedogenic Fe oxi-hydroxides to pseudo-total Fe ratio. The microbial biomass content and structure, and the enzyme activities significantly differed in the four pedological layers. The amount of microbial biomass was, as expected, most abundant in the Tp layer, where bacteria and actinomycetes abounded. Conversely, in Elu and Wh we observed a fungal-to-bacterial biomass ratio significantly higher than in Tp and Ls; in Elu, also the gram (+)/gram (−) ratio was the highest. In the upper layer, the interaction between enzymes and minerals like disordered smectite and pedogenic Fe-oxides appeared as responsible for the inhibition of the total enzyme activity per unit of organic C, and of the lipase activity. In Ls layer, where clay illuviation and high organo-minerals interaction occurred, the potential hydrolysis of organic matter was low, as revealed by the SEI/TOC ratio, the reduced lipase activity, and the inhibited activity of α-fucosidase and α-mannosidase. Even if the activity of most enzymes depends on the substrate availability, which decreases with soil depth, those involved in lipid degradation displayed the maximum activities in Elu and Wh layers, where a relative increase of the fungal population was observed. In conclusion, our findings showed that the soil functionality, expressed by the microbial community structure and enzymes activity, can vary according to organic matter–mineral interaction following the weathering and lessivage gradients along the soil profiles. • The effects of weathering and lessivage on soil biochemical properties were assessed. • Six soil profiles were examined in North-eastern Italian Alps (Brocon Pass 1600 m a.s.l.). • Soil functionality expressed by microbial activity vary following weathering and lessivage. • In Bw horizons the microbial functions related to SOM degradation is more conservative. • In Bt horizons C sequestration could become fulfilled. [ABSTRACT FROM AUTHOR]