TNF-alpha and lymphotoxin (LT, TNF-beta) genes are tandemly arranged and map within the MHC centromeric to HLA-B and telomeric to the class III genes. Both cytokines encoded by these genes are potent immunomodulators. On the other hand, some MHC-linked autoimmune diseases are characterized by abnormal levels of their expression or inducibility. A search for the putative disease-associated TNF/LT alleles depends on the informative genetic markers at the TNF locus. Previously, a low degree of genetic polymorphism at the human TNF locus has been reported, mostly bi-allelic RFLP. To localize and define additional polymorphic markers, we probed the collection of genomic clones with synthetic tandemly repeated dinucleotides, corresponding to the sequences known as microsatellites. We mapped and characterized three (TC/GA) and one (AC/GT) repeats within cloned 40-kb DNA comprising the human TNF locus. Using a polymerase chain reaction-based technique, we analyzed three of these four microsatellites and observed their length of polymorphism. Using DNA samples from blood donors, two families, and three human cell lines, we detected 13 distinct alleles of the AC/GT microsatellite neighboring human TNF genes. The variability was further increased by simultaneous analysis of the second linked microsatellite. This linked TC/GA repeat showed at least five alleles, whereas the least polymorphic TC/GA repeat located in the first intron of LT (TNF-beta) gene had two alleles. TNF alleles defined by microsatellites were stably inherited and segregated in the Mendelian way. Therefore, we describe thus far the most informative level of DNA sequence polymorphism in this part of human MHC. We propose a nomenclature for microsatellite tagged LT/TNF alleles based on their size and variability, which could also be extended to include RFLP and other not yet identified polymorphic markers. Microsatellite tagged polymorphism described here can be used in systematic linkage studies of HLA-associated diseases.