Transcription regulatory elements have been analyzed in upstream sequences of an Interleukin-6 (II-6) primary response gene, MyD88. MyD88 2.3 kb mRNA is strongly and persistently induced in the course of myeloleukemic M1 cells differentiation with II-6. MyD88 cDNA sequences were found in a region of 12 kb of mouse genomic DNA. Using II-6 treated M1 cell RNAs, two transcription start sites have been localized, ∼100 bp upstream from the 5′ end of the cloned cDNA. We sequenced 1.4 kb of 5′ genomic DNA including the first exon. In 5′ of mRNA transcription start site, MyD88 nucleotidic sequence is 85% identical to 5′ complementary sequences of the rat 3′-ketoacetyl CoA thiolase gene, over 1.2 kb. A DNA element conferring II-6-inducible transcription to reporter genes, and localized 30 bp upstream of MyD88 first RNA start site, contains overlapping binding sites for cytokine activated transcription factors Stat and for the Interferon Regulatory Factor-1 and -2 (IRF-1 and IRF-2). In vitro binding assays showed that attachment of Stat factors to this element early in II-6 treatment requires tyrosine kinase activation. IRF1, an activator of transcription, is also induced to bind to this sequence at later times. A model of persistent activation of MyD88 gene through these two types of factors is proposed.