Multiple tracers were pumped into upper basement around Hole U1362B during Integrated Ocean Drilling Program (IODP) Expedition 327 as part of a single- and cross-hole tracer experiment on the eastern flank of Juan de Fuca Ridge. Tracers injected were sulfur hexafluoride (dissolved gas), cesium chloride hexahydrate, erbium chloride, and holmium chloride hexahydrate (solutes), and several sizes of fluorescent microspheres and fluorescent-stained microbes filtered from surface seawater (particles). Tracers were injected as part of a 24 h pumping experiment intended to test a large volume of basement rock around Hole U1362B. We report on the design of the tracer experiment, methods used to prepare and inject tracers using shipboard mud and cement pump systems, and tools developed to permit shipboard and downhole sampling of injectate. Shore-based analysis of injectate samples will be essential for interpretation of long-term samples collected from subseafloor borehole observatories (“CORKs”). Borehole samples are being collected continuously within a long-term CORK installed in Hole U1362B after tracer injection was complete and within similar CORK systems installed in nearby boreholes before and during Expedition 327. CORK servicing expeditions are currently planned for summer 2011 and 2012. These expeditions and additional work in subsequent years will provide data and samples that will permit a quantitative assessment of tracer transport behavior in the upper ocean crust.