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 clone WP-3.27 produces an antigen-specific factor, OX-F, that acts in an Ag-specific manner to initiate DTH. The clone was phenotyped as a Thy-1+, B220+, CD3-, CD4-, CD8- cell. In this report, we further detail the characteristics of this unusual Ag-specific DTH-initiating cell clone. By flow cytometry analysis, WP-3.27 is Thy-1lo, Lyt-1+ (CD5+), but CD3-, TCR-alpha beta-, and TCR-gamma delta-. Moreover, WP-3.27 does not express surface immunoglobulins but expresses B220 (CD45RA), and also some macrophage markers such as Mac-1, F4-80, and MHC class II after gamma-IFN treatment. Interestingly, this clone also expresses IL-3 receptors (IL-3R) and not IL-2R. In addition to the Ag-specific DTH-initiating factor, WP-3.27 constitutively produces IL-3. Inhibition of proliferation of WP-3.27 with an anti-mouse IL-3 monoclonal antibody suggests that the clone WP-3.27 is IL-3-dependent, at least partially. WP-3.27 also constitutively produces IL-1 and IL-6, but not TNF-alpha. LPS activation of the clone resulted in a net increase of IL-1, IL-6, and TNF-alpha production. Thus, this Ag-specific DTH-initiating cell clone makes a unique set of cytokines. Northern blot analysis demonstrated that clone WP-3.27 transcribes mRNA encoding IL-1, IL-3, IL-6, and TNF-alpha, but not for TNF-beta (lymphotoxin). The nature of this unusual cell, which displays characteristics of more than one cell lineage, is discussed.