Abandonment of agricultural land and the subsequent recolonization by natural vegetation is known to cause increases in C contents, contributing to reduction in atmospheric CO 2 concentrations. Assessment of the possible mitigation of CO 2 excess requires understanding the SOC dynamics, the origin of C pools and the pathways of their transformation. The aims of this work were to assess, by using the δ 13 C signature, the changes of old and new organic C in total (soil organic carbon, SOC) and labile (microbial biomass C, MBC, dissolved organic C, DOC, CO 2 efflux from soil) pools after vegetation change from vineyard (C 3 ) to grassland (C 4 ) under semiarid Mediterranean climate. Colonization of abandoned vineyard by the perennial C 4 -grass Hyparrhenia hirta after 15 or 35years increased topsoil C stocks by 13% and 16%, respectively. Such an increase was attributed to new above- and below-ground biomass C input from H. hirta. The maximal incorporation of new C was observed in MBC, whereas the DOC derived mainly from old SOC. Based on δ 13 C values of SOC, MBC, DOC and CO 2 in C 3 soil and in soils after 15 and 35years of C 4 grass colonization, 13 C fractionation per se from changes in isotopic composition by preferential utilization of substrates with different availability was separated. MBC in C 3 –C 4 soil used more recent ( 13 C-enriched) versus old C (relatively 13 C-depleted) sources. The Δ 13 C by decomposition of SOC to CO 2 (δ 13 C of CO 2 minus δ 13 C of SOC) was higher than Δ 13 C by microbial respiration (CO 2 minus MBC), demonstrating that under semiarid climate, soil microorganisms do not always preferentially decompose the most available SOC pools. The use of δ 13 C signature of SOC after C 3 –C 4 vegetation change combined with soil incubation is a powerful tool to assess the exchange between old and new C in pools of various availability.