Background Cystatin F is a protein inhibitor of cysteine peptidases, expressed predominantly in immune cells and localised in endosomal/lysosomal compartments. In cytotoxic immune cells cystatin F inhibits both the major pro-granzyme convertases, cathepsins C and H that activate granzymes, and cathepsin L, that acts as perforin activator. Since perforin and granzymes are crucial molecules for target cell killing by cytotoxic lymphocytes, defects in the activation of either granzymes or perforin can affect their cytotoxic potential. Materials and methods Levels of cystatin F were assessed by western blot and interactions of cystatin F with cathepsins C, H and L were analysed by immunoprecipitation and confocal microscopy. In TALL-104 cells specific activities of the cathepsins and granzyme B were determined using peptide substrates. Results Two models of reduced T cell cytotoxicity of TALL-104 cell line were established, either by treatment by ionomycin or by immunosuppressive transforming growth factor beta. Reduced cytotoxicity correlated with increased levels of cystatin F and with attenuated activities of cathepsins C, H and L and of granzyme B. Co-localisation of cystatin F and cathepsins C, H and L and interactions between cystatin F and cathepsins C and H were demonstrated. Conclusions Cystatin F is designated as a possible regulator of T cell cytotoxicity, similar to its role in natural killer cells.