The mouse CD40 ligand (CD40L) gene was cloned, sequenced and characterized. DNA sequence analysis showed that the CD40L gene comprises five exons and four intervening introns, spread over 13-14 kb of genomic DNA. The putative site for initiation of mRNA transcription was identified at 67 bp upstream of the translation initiation (ATG) codon. The nucleotide sequence of the 5'-flanking region of this gene revealed the presence of several regulatory regions including a TATA-like box, an Sp1-like box and six potential NF-AT-like motifs. The 3'-untranslated region of the murine CD40L gene contained two ATTTA-elements which are thought to confer instability to the mRNA of many cytokines and two adjacent dinucleotide repeates, (CT)25 and (CA)45. These elements may play a role in the post-transcriptional regulation of CD40L gene expression.