The gamma interferon (IFN-␥) enzyme-linked immunospot (ELISPOT) assay is used routinely to evaluate the potency of human immunodeficiency virus (HIV) vaccine candidates and other vaccine candidates. In orderto compare candidates and pool data from multiple trial laboratories, validated standardized methods must be applied across laboratories. Proficiency panels are a key part of a comprehensive quality assurance program to monitor inter-and intralaboratory performance, as well as assay performance, over time. Seven International AIDS Vaccine Initiative-sponsored trial sites participated in the proficiency panels described in this study. At each laboratory, two operators independently processed identical sample sets consisting of frozen peripheral blood mononuclear cell (PBMC) samples from different donors by using four blind stimuli. PBMC recovery and viability after overnight resting and the IFN-␥ ELISPOT assay performance were assessed. All sites demonstrated good performance in PBMC thawing and resting, with a median recovery of 78% and median viability of 95%. The laboratories were able to detect similar antigen-specific T-cell responses, ranging from 50 to >3,000 spot-forming cells per million PBMC. An approximate range of a half log in results from operators within or across sites was seen in comparisons of antigen-specific responses. Consistently low background responses were seen in all laboratories. The results of these proficiency panels demonstrate the ability of seven laboratories, located across three continents, to process PBMC samples and to rank volunteers with differential magnitudes of IFN-␥ ELISPOT responses. These findings also illustrate the ability to standardize the IFN-␥ ELISPOT assay across multiple laboratories when common training methods, reagents such as fetal calf serum, and standard operating procedures are adopted. These results are encouraging for laboratories that are using cell-based immunology assays to test HIV vaccines and other vaccines.Most human immunodeficiency virus (HIV) vaccines currently in development aim to induce cellular immune responses, since these have been shown previously to temporally correlate with the containment of virus in infected individuals and, more significantly, to be crucial in the suppression of virus in the rhesus macaque model (2,13,15,25). The ability to measure and quantitate cellular immune responses has been facilitated through the development of enzyme-linked immunospot (ELISPOT) and flow cytometry assays which determine the number of antigen-specific cells through surrogate markers of effector function, such as cytokine production or the degranulation of lytic granules (1,8,23,29), and are more quantitative and less labor-intensive than traditional assays that detect T-cell responses, such as 51 Cr release and lymphoproliferation assays (19). The gamma interferon (IFN-␥) ELISPOT assay is a primary assay employed to measure vaccine immunogenicity in HIV vaccine clinical trials, in addition to trials in the cancer, malaria, and tuberculosi...