Human lymphotoxin-alpha (LT alpha) is found in a secreted form and on the surface of lymphocytes as a complex with a second related protein called lymphotoxin-beta (LT beta). Both secreted human LT alpha and TNF have similar biological activities mediated via the TNF receptors, whereas the cell surface LT alpha beta complex binds to a separate receptor called the LT beta receptor (LT beta R). The murine LT alpha and LT beta (mLT alpha and mLT beta) proteins have never been characterized. When recombinant mLT alpha was produced by either of several methods, the protein had a very low specific activity relative to that of human LT alpha in the conventional WEHI 164 cytotoxicity bioassay. The weak activity observed was inhibited by a soluble murine TNF-R55 Ig fusion protein (mTNF-R55-Ig), but not by mLT beta R-Ig. Coexpression of both mLT alpha and a soluble version of mLT beta in insect cells led to an LT alpha beta form that was cytotoxic in the WEHI 164 assay via the LT beta R. To determine whether natural mLT alpha-like forms with cytotoxic activity comparable to that of secreted human LT alpha were secreted from primary spleen cells, splenic lymphocytes were activated in various ways, and their supernatants were analyzed for cytotoxic activity. Using specific Abs to distinguish between mTNF and mLT, a TNF component was readily detected; however, there was no evidence for a secreted mLT alpha cytotoxic activity using this assay. Combined, these observations suggest that secreted mLT alpha may not play a role in the mouse via interactions with TNF-R55, and the ramifications of this hypothesis are discussed.