Lymphotoxin alpha (LTalpha) can exist in soluble form and exert tumor necrosis factor (TNF)-like activity through TNF receptors. Based on the phenotypes of knockout (KO) mice, the physiological functions of LTalpha and TNF are considered partly redundant, in particular, in supporting the microarchitecture of the spleen and in host defense. We exploited Cre-LoxP technology to generate a novel neomycin resistance gene (neo) cassette-free LTalpha-deficient mouse strain (neo-free LTalpha KO [LTalphaDelta/Delta]). Unlike the "conventional" LTalpha-/- mice, new LTalphaDelta/Delta animals were capable of producing normal levels of systemic TNF upon lipopolysaccharide (LPS) challenge and were susceptible to LPS/D-galactosamine (D-GalN) toxicity. Activated neutrophils, monocytes, and macrophages from LTalphaDelta/Delta mice expressed TNF normally at both the mRNA and protein levels as opposed to conventional LTalpha KO mice, which showed substantial decreases in TNF. Additionally, the spleens of the neo-free LTalpha KO mice displayed several features resembling those of LTbeta KO mice rather than conventional LTalpha KO animals. The phenotype of the new LTalphaDelta/Delta mice indicates that LTalpha plays a smaller role in lymphoid organ maintenance than previously thought and has no direct role in the regulation of TNF expression.