The tumor necrosis factor-alpha (TNF-alpha, or TNF) genes of NOD mice and NZW mice are reportedly underexpressed relative to the TNF genes of control mice in lipopolysaccharide (LPS)-induced peritoneal macrophages. These findings, as well as the well-known major histocompatibility complex (MHC) linkage of the TNF genes, have prompted speculation that mutations affecting expression of the TNF-alpha loci might represent a primary cause of autoimmune diseases. Differences in expression of the TNF genes in different strains of mice might result either from effects of cis-acting mutations or from differences in the cellular environment that operate in trans. To discriminate between these possibilities, we directly examined the relative contribution made by each of two different TNF alleles (one associated with an autoimmune-prone haplotype and the other not) to the pool of TNF mRNA within the cells of F1 hybrid mice. Reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify a polymorphic fragment derived from the total pool of TNF mRNA present in LPS-induced peritoneal macrophages of hybrid animals produced by crossing BALB/c to non-obese diabetic (NOD), and New Zealand black (NZB) to New Zealand white (NZW). In both types of F1 hybrid, the two alleles were represented in nearly equal quantities at the mRNA level. It may be inferred that at all pretranslational levels, the NZW and NOD TNF alleles are functionally equivalent to the control alleles that were examined. Interstrain differences in responsiveness to LPS are therefore responsible for interstrain differences in TNF gene expression.