Cysteine (Cys) and methionine (Met) are central to terrestrial S cycling because they are sources of carbon (C), nitrogen (N), and sulphur (S) for plant nutrition and microbial growth. However, soil microorganisms are expected to compete for the C, N and S in these S-amino acids. We hypothesized that microbial competition would be greater in soils with low plant productivity due to lower C inputs from plants. Here we added 14C-labelled Cys and Met to 5 soils collected from an altitude-driven primary grassland productivity gradient, we then measured microbial uptake with a centrifugal drainage procedure over 60 min, and the subsequent mineralization with NaOH traps over 48 h. Our results revealed that both Cys and Met were rapidly assimilated by soil microbes, with half-lives ranging from 0.34 to 2.14 min, which is an order of magnitude (or more) faster than when determined from measurement of 14CO2 evolution. This considerable delay between microbial 14C removal from soil solution and subsequent 14CO2 evolution indicates that the degradation of Cys and Met in grassland soils occurred mainly through biological processes. Soil microbial uptake of Cys and Met was dominated by a high-affinity transport system (0.01–0.1 mM), while a lower affinity transport system became more important at higher substrate concentrations (1–100 mM). In addition, microbial uptake and mineralization rates of Cys and Met declined in less productive, higher elevation sites, suggesting that the turnover of organic N and S, and subsequent availability for plant uptake is likely to be controlled by soil fertility. We conclude that although Cys and Met may represent a minor component of DON and DOS pools in soil, their importance for soil microbes and plant nutrition may have been underestimated due to their fast turnover and replenishment rates in grassland soils.