Although the requirements for T lymphocyte homing to lymph nodes (LNs) are well studied, much less is known about the requirements for T lymphocyte locomotion within LNs. Imaging of murine T lymphocyte migration in explanted LNs using two-photon laser-scanning fluorescence microscopy provides an opportunity to systematically study these requirements. We have developed a closed system for imaging an intact LN with controlled temperature, oxygenation, and perfusion rate. Naive T lymphocyte locomotion in the deep paracortex of the LN required a perfusion rate of> 13 μm/s and a partial pressure of O 2 (pO 2) of> 7.4%. Naive T lymphocyte locomotion in the subcapsular region was 38% slower and had higher turning angles and arrest coefficients than naive T lymphocytes in the deep paracortex. T lymphocyte activation decreased the requirement for pO 2, but also decreased the speed of locomotion in the deep paracortex. Although CCR7−/− naive T cells displayed a small reduction in locomotion, systemic treatment with pertussis toxin reduced naive T lymphocyte speed by 59%, indicating a contribution of Gα i-mediated signaling, but involvement of other G protein-coupled receptors besides CCR7. Receptor knockouts or pharmacological inhibition in the adenosine, PG/lipoxygenase, lysophosphatidylcholine, and sphingosine-1-phosphate pathways did not individually alter naive T cell migration. These data implicate pO 2, tissue architecture, and G-protein coupled receptor signaling in regulation of naive T lymphocyte migration in explanted LNs.