ABSTRACT Identification of interventions that extend mouse lifespan provides new insights into mechanisms of longevity determination in mammals, and may lay the groundwork for eventual anti-aging therapies in humans. The NIA Interventions Testing Program (ITP) evaluates agents proposed to extend mouse lifespan by retardation of aging or postponement of late life diseases. Interventions proposed by multiple collaborating scientists from the research community are tested, in parallel, at three sites (Jackson Laboratories, University of Michigan and University of Texas), using very similar, standardized protocols, and using sufficient numbers of genetically heterogeneous mice to provide 80% power for detecting changes in lifespan of 10%, for either sex, after pooling data from any two of the test sites. One hundred and two such lifespan experiments, involving various doses of 66 distinct agents, have been initiated in the first twenty years of the ITP. Thirty-six experiments have involved comparative tests of multiple doses of effective agents, variable starting ages, or alternative dosing schedules. Statistically significant effects on longevity, in one or both sexes, have been documented and then confirmed for NDGA, rapamycin, acarbose, 17-α-estradiol (17aE2), and canagliflozin. Significant effects were also noted for Protandim, glycine, meclizine, captopril, and astaxanthin. Lifespan trials are now underway for 25 new agents. ITP survival results have also documented longevity benefits from four agents started in middle-age: rapamycin, acarbose, 17aE2, and canagliflozin. A Collaborative Interactions Program (CIP) has provided tissues from ITP drug-treated mice to an open, growing, international network of scientific collaborators, meeting 26 requests from 17 distinct laboratories in the previous five-year period. Plans for the next five-year period include additional lifespan ("Stage 1") trials, detailed analyses ("Stage 2") of agents found to increase lifespan, transition from the CIP to an Interventions Biospecimens Repository, additional diagnostic specificity in pathological assessments, inclusion of RNA-Seq data in all Stage 2 protocols, and comprehensive pharmacokinetic assessments of drugs found to increase lifespan, as well as continued collaborative work with a network of scientists to study drug effects on postulated aging mechanisms and links to disease. Site-specific studies at Michigan will add a battery of tests for Aging Rate Indicators in fat, muscle, brain, plasma, and liver of Stage 2 mice, and include tests of drug effects on learning and memory in Stage 2 protocols. The work proposed should allow the ITP to continue to make major contributions to mammalian aging biology.