Interleukin-6 (IL-6) is a multifactorial cytokine produced by many cells including monocytes and macrophages in the immune-stimulated host. We measured IL-6 activity induced by muramyl dipeptide (MDP) and lipopolysaccharide (LPS) in vitro and by liposome-encapsulated muramyl tripeptide-phosphatidylethanolamine (L-MTP-PE) in vivo in normal dogs. Adherent mononuclear cells were cultured with MDP, LPS, or MDP plus LPS for various time periods. After incubation, culture supernatants were collected and assayed for IL-6 activity. Sera from dogs following L-MTP-PE administration were also evaluated for IL-6 activity. IL-6 activity both in supernatants and sera was measured using a 7TD1 bioassay. Significantly elevated IL-6 activity could be measured as early as 2 hours after mononuclear cells were exposed to MDP, LPS, or MDP plus LPS. IL-6 activity induced by LPS was greater than that induced by MDP, and the combination of MDP and LPS induced the greatest increase in IL-6 activity. Serum IL-6 activity was elevated within 3 to 4 hours post L-MTP-PE administration and subsequently declined to pretreatment level at 24 hours post injection. Neutralization of supernatant and serum IL-6 activity was not achieved with goat or rabbit anti-recombinant human IL-6 polyclonal antibody. This study demonstrates that MDP and LPS, alone and in combination, can induce enhanced IL-6 activity of canine adherent mononuclear cells in vitro, and that intravenous injection of L-MTP-PE is capable of eliciting increased IL-6 activity in vivo in normal dogs. These findings suggest that IL-6 may play an important role in the biologic response observed in canine cancer patients treated with L-MTP-PE.