Abstract : Soil microorganisms can control the soil cycles of carbon (C), and depending on their C-use efficiency (CUE), these microorganisms either contribute to C stabilization in soil or produce CO2 when decomposing organic matter. However, little is known regarding the enzyme investment of microbial decomposers and the effects on their CUE. Our objective was to elucidate the strategies of litter-decomposing fungi as a function of litter quality. Fungal biosynthesis and respiration were accounted for by quantifying the investment in enzyme synthesis and enzyme efficiency. The basidiomycete Phanerochaete chrysosporium was grown on the leaves, stems, and roots of maize over 126 days in controlled conditions. We periodically measured the fungal biomass, enzyme activity, and chemical composition of the remaining litter and continuously measured the evolved C–CO2. The CUE observed for the maize litter was highest in the leaves (0.63), intermediate in the roots (0.40), and lowest in the stems (0.38). However, the enzyme efficiency and investment in enzyme synthesis did not follow the same pattern. The amount of litter C decomposed per mole of C-acquiring hydrolase activity was 354 µg C in the leaves, 246 µg C in the roots, and 1541 µg C in the stems (enzyme efficiency: stems > leaves > roots). The fungus exhibited the highest investment in C-acquiring enzyme when grown on the roots and produced 40–80% less enzyme activity when grown on the stems and leaves (investment in enzymes: roots > leaves > stems). The CUE was dependent on the initial availability and replenishment of the soluble substrate fraction with the degradation products. The production of these compounds was either limited because of the low enzyme efficiency, which occurred in the roots, or because of the low investments in enzyme synthesis, which occurred in the stems. Fungal biosynthesis relied on the ability of the fungus to invest in enzyme synthesis and the efficient interactions between the enzymes and the substrate. The investment decreased when N was limited, whereas the efficiency of the C-acquiring enzymes was primarily explained by the hemicellulose content and its embedment in recalcitrant lignin linkages. Our results are crucial for modeling microbial allocation strategies.