The elicitation of delayed-type hypersensitivity (DTH) reactions in mice is due to the sequential action of two different, antigen-specific, Thy-1+ cells. We have previously cloned the early-acting DTH-initiating cell from nude mice that were immunized and boosted by contact sensitization with oxazolone (OX). This cell clone, WP-3.27, releases an antigen-specific factor (OX-F) that sensitizes mast cells such that specific antigen challenge will induce serotonin release which mediates the early phase of DTH. In normal mice contact sensitized with picryl chloride (PCl), a similar polyclonal factor (PCl-F) has a similar activity and is also known to bind to macrophages. Thus, we measured macrophage production of TNF alpha, IL-1, IL-6, and PGE2 in response to the hapten affinity-purified DTH-initiating factors OX-F and PCl-F. Both factors induced significant release of each cytokine and PGE2. The production of TNF alpha, IL-1, and IL-6 was measured by bioassays. Northern blot analysis showed rapid accumulation of cytokine mRNA (2-4 hr), while maximal production of PGE2 occurred at approximately 8 hr. These macrophage activating properties of OX-F and PCl-F were not due to contamination with LPS as determined by the low levels of LPS present in OX-F and PCl-F and by the failure of polymyxin B to inhibit factor-induced PGE2 and TNF alpha production. Also, macrophage activation was shown not to be due to the action of several lymphokines known to be produced by WP3.27. Separation of OX-F and PCl-F by preparative isoelectric focusing showed a similar pattern: there were two major peaks of PGE2-inducing activity observed for both factors (for PCl-F at pI of 2-3 and 5.0, and for OX-F at pI of 3.5-4 and 5.0), but not for a sham factor produced by WEHI-3 cells. The ability of DTH-initiating factors to rapidly induce macrophage cytokine release and PGE2 synthesis 4-6 hr later may suggest a role for these mediators during the respective early vascular and late cellular phases of inflammation in DTH.