Papain family cysteine proteinases have long been associated with lysosomal and phagosomal compartments. Aqueous extracts of Tetrahymena contain a variety of proteinases, predominantly papain family cysteine proteinases sensitive to E64 and other broad spectrum cysteine proteinase inhibitors. While there are 114 candidate cysteine proteinase genes in the Tetrahymena genome, few have been characterized with regard to expression, localization, and function. Membrane permeable and non-permeable fluorogenic proteinase substrates and fluorescent inhibitors localize primarily to small (≤1 µm) vesicles, usually on the surface or interior of phagosomes. Non-permeable fluorogenic substrates are taken up through the oral apparatus and incorporated into vesicles that initially align with the postoral microtubule system (detected by antibody staining), and subsequently with discrete phagosomes in transit toward the cell posterior. Zymographic analyses of aqueous cell extracts reveal 11 - 16 discrete cysteine proteinases ranging in size from about 25 kDa to over 150 kDa. A proteomic approach was used to determine which of the 114 candidate proteinases were present in aqueous and detergent homogenates. Briefly, homogenates were partially purified by affinity chromatography against immobilized inhibitor, then subjected to reduction, alkylation, and trypsin digestion.. Resulting peptides were further purified by HPLC coupled to tandem mass spectrometry and SpectrumMill analytical software. We identified 12 valid papain family cysteine proteinase sequences were detected. Future efforts will focus on additional proteomics as well as determining cellular localization, function, and gene structure of these enzymes.