A single intradermal injection of the adjuvant-oil squalene induces T cell-mediated arthritis in DA rats. The chain of events leading from non-specific provocation of the immune system to arthritis, with clinical similarities to rheumatoid arthritis, is largely undetermined. Here, we combined in vivo tracking of tritium-labelled squalene with lymph node (LN) cell transfer experiments to determine where critical activation events may take place. The majority of squalene remained at the injection site (79%). The amounts recovered in peripheral joints (<1%) were equal to that recovered in other organs that can be targets in autoimmune diseases. This argues that arthritis does not develop as a consequence of adjuvant accumulation in joints. In contrast, substantial amounts of squalene were recovered in hyperplastic LN draining the injection site (1–13%). The adjuvant was deposited to a larger extent in cells than in extracellular matrix. The draining LN cells could transfer arthritis to naïve irradiated DA rats following in vitro stimulation with conA. Interestingly, non-draining LN were also hyperplastic and harboured arthritogenic cells, although they contained low amounts of squalene (<1%). Consequently, the amount of arthritogenic adjuvant in a particular LN is not closely linked to the development of pathogenic cells. The distribution pattern of squalene was similar in MHC-identical but arthritis-resistant PVG.1AV1 and LEW.1AV1 rats, and it was unaffected by T cell depletion with a monoclonal antibody (R73). Thus, T cells and non-MHC genes do not regulate dissemination of squalene, but rather determine arthritis development at the level of adjuvant response.