p36 (also termed annexin II) is a 39 kDa Ca2+/phospholipid-binding, membrane-associated protein that is a protein-tyrosine kinase substrate. We report here studies of the noncoding exons of p36, which combined with our earlier studies of the coding exons, allow us to conclude that the murine p36 gene is 34 kb in length with 14 exons. Comparison of the genes coding for mouse and human p36 (annexin II) and mouse, rat and human p35 (annexin I) and pigeon cp35 (an annexin I-related protein) shows strong genomic structural conservation supporting the hypothesis that these genes had a common ancestor. Both human and murine p36 mRNAs were found to be alternatively spliced in their 5' noncoding region. In both cases exon 2 is a cassette exon, which is present in a small fraction of p36 mRNAs. In type 1 mouse p36 mRNA the first noncoding 44 base exon 1 is joined to exon 3, the first of the 12 coding exons. In type 2 mRNA a 70 base noncoding exon (exon 2) is inserted between exon 1 and exon 3. Type 1 mRNA was present in all cell types studied as revealed by Northern analysis and primer extension, whereas type 2 mRNA could only be detected by RACE or PCR, indicating that it is of very low abundance. The major transcription start site of the mouse p36 gene was mapped by primer extension to be 61 bp upstream of the AUG initiation codon, which corresponds to type 1 mRNA, The murine p36 gene enhancer/promoter region contains a putative TATA box and several other potential regulatory sequences. The two alternatively-spliced human p36 mRNAs differ by the presence or absence of a noncoding 81 base exon (exon 2) inserted after exon 1, with exon 2-containing mRNAs representing approximately 10% of total p36 mRNA. The 300 bp spanning the promoter and exons 1-3 of the human and murine p36 genes show strong sequence homology immediately before and after the major transcription start site except in the region corresponding to exon 2, where homology is more limited.