The >1 kb XL-exon of the rat XLalphas/Galphas gene encodes the 37 kDa XL-domain, the N-terminal half of the 78 kDa neuroendocrine-specific G-protein alpha-subunit XLalphas. Here, we describe a novel feature of the XL-exon, the presence of an alternative >1 kb open reading frame (ORF) that completely overlaps with the ORF encoding the XL-domain. The alternative ORF starts 32 nucleotides downstream of the start codon for the XL-domain and is terminated by a stop codon exactly at the end of the XL-exon. The alternative ORF encodes ALEX, a very basic (pI 11.8), proline-rich protein of 356 amino acids. Both XLalphas and ALEX are translated from the same mRNA. Like XLalphas, ALEX is expressed in neuroendocrine cells and tightly associated with the cytoplasmic leaflet of the plasma membrane. Remarkably, ALEX binds to the XL-domain of XLalphas. Our results reveal a mechanism of gene usage that is without precedent in mammalian genomes.