Cryopreserved peripheral blood mononuclear cells (PBMC) are a good alternative to fresh PBMC in clinical trials when samples are collected at multiple sites and processed later. Optimal retention of cryopreserved PBMC functionality depends on cell isolation, freezing and storage. SeraCare developed validated PBMC cryopreservation procedures to ensure functionality is maintained on cryopreservation. Two assays were performed to demonstrate the equivalence of cryopreserved vs. fresh PBMCs. Intracellular cytokine staining (ICS) quantitatively measures antigen specific T cell cytokine production at the single cell level. Using flow cytometry ICS analyzes multiple parameters on a single sample to obtain the precise cytokine producing T cell phenotype. Our data demonstrates production of IFN-γ, IL-2, TNF-α and IL-4 from cryopreserved PBMC suggesting full retention of PBMC functionality after cryopreservation. The antibody dependent cellular cytotoxicity (ADCC) assay measures natural killer cell mediated destruction of antibody targeted cells. ADCC is a powerful tool to measure immune responses in HIV and cancer immunotherapy. Most studies used fresh PBMC in ADCC assays, noting that cryopreserved PBMC result in lower activity. We show cryopreserved PBMCs have comparable ADCC activity to fresh PBMC. ADCC activity appeared donor specific and was not affected by cryopreservation. Our data suggest that cryopreservation has no adverse affect on PBMC functionality in immunological assays.