The MR1-MAIT cell axis plays important roles in health and disease, but its study is hampered by the lack of activating and inhibitory ligands that are both stable and potent. Here, we report new water stable ligands with distinctive functions and potent activities. We developed a new stable antigen that activates the MAIT TCR with comparable picomolar potency to the microbial antigen 5-OP-RU. We also developed the most potent inhibitor of MAIT TCR activation to date (nanomolar IC50), surpassing the potency and solubility of established inhibitor Ac-6-FP. Further, we report new MR1 ligands that enable protein-pulldown and imaging applications, highlighting a strategy for creating custom probes to explore MR1-dependent cell biology. Supported by computer modelling, our ligands shed new light on the molecular basis of MR1 folding and MAIT TCR activation potency, and may inform the design of future MR1-binding therapeutics.