Major histocompatibility complex (MHC) class I molecules display peptides on cell surfaces for subsequent T-cell recognition and are involved in the immune response against intracellular pathogens. In this study, a BAC library was created from a single three-spined stickleback and screened for clones containing MHC class I genes. In a 163.2-kb genomic sequence segment of a single clone, we identified three MHC class I genes in the same transcriptional orientation. Two class I genes are potentially expressed and functional. In one class I gene, the transmembrane region is missing and could therefore present a pseudogene. Alternatively, it presents a functional gene that encodes a soluble MHC class Ib molecule. Despite genomic similarities to the MHC class II region, which is characterized by interlocus recombination, we did not find any evidence for this kind of recombination in the class I genes. It thus seems that interlocus recombination may play a rather minor role in generating class I diversity in stickleback and that the class I region displays a higher genomic stability (i.e., lower local recombination rate). In addition, two non-MHC genes (Oct-2 beta and Na+,K+-ATPaseα3) have been identified in the analyzed class I region. The Oct-2 beta gene is a transcription factor that is expressed primarily in B lymphocytes, in activated T-cells, and in neuronal cells. The Na+,K+-ATPaseα3 gene is primarily expressed in the brain and heart and mediates catalytic activities. Both genes are located on the same linkage group together with the MHC class I genes in the zebra fish. In humans, however, homologues of Oct-2 beta and ATPaseα3 lie outside the MHC region, which indicates that the concentration of immune genes found in mammalian genomes is a derived state. © 2007 Springer-Verlag.