Macrophage activation factor (MAF) activity, assessed by the ability to activate macrophages (MO) to lyse RBL--a TNF-resistant, retrovirally transformed, tumor target--was detected in the PHA-stimulated supernatant (Sup) of LBRM, a murine T cell line. LBRM Sup provided a priming signal to MO, but required the subsequent addition of small amounts of LPS for the expression of tumor cytotoxicity. The identity of the lymphokine responsible for this MAF activity was investigated. IFN-gamma, the only previously characterized lymphokine capable of priming MO for tumor cytotoxicity, did have MAF activity in the assay, but IFN-gamma could not be detected by ELISA in LBRM Sup, and LBRM-derived mRNA lacked detectable message for IFN-gamma. Moreover, anti-IFN-gamma failed to inhibit the MAF activity of LBRM Sup, suggesting that the presence of small, undetectable amounts of IFN-gamma were neither responsible nor required for LBRM MAF activity. LBRM MAF activity appeared distinct from the other previously identified lymphokines produced by LBRM, since granulocyte-macrophage-CSF, IL-2, and IL-3 purified from LBRM Sup were unable to activate MO to lyse RBL. IL-4 and TNF, two lymphokines not known to be produced by LBRM but able to activate MO for cytotoxicity of some tumor targets, were also unable to activate MO for RBL cytotoxicity. LBRM MAF lacked antiviral activity in biologic assays, further distinguishing the lymphokine from IFN-gamma, and had an apparent Mr of 30,000 Da using gel filtration chromatography. Thus, the LBRM T cell line produces a previously undescribed lymphokine that primes MO for tumor cytotoxicity.