MF59, which is an adjuvant approved for human use, typically elicits higher antibody titers than alum when used in combination with a variety of recombinant and natural subunit antigens. The mechanisms responsible for the adjuvant action of MF59 are not fully understood. In particular, little is known about the in vivo distribution of MF59 and of antigen after intramuscular (i.m.) injection. The goal of the present study was to determine the distribution of MF59 injected with soluble antigen gD2 from type 2 herpes simplex virus (HSV) and to compare the distribution of gD2 injected with or without MF59. At 4 h, 36% of the injected dose of labeled MF59 was in the quadriceps muscle and about 50% was in the inguinal fat surrounding the muscle. Half of the initial amount of labeled MF59 in muscle was detected 42 h after injection. The amount of labeled MF59 in the draining lymph nodes was maximal 2 d after injection, which represented 0.1-0.3% of the injected dose. At 4 h, 12% of the injected dose of labeled gD2 was found in the muscle. The presence of MF59 did not significantly modify the distribution of gD2. The results indicate that MF59 and gD2 distribute and are cleared independently after i.m. injection. Importantly, MF59 is unlikely to have a repository effect, whereby it slowly releases the antigen.