Four cDNAs encoding the major histocompatibility complex (MHC) class I alpha chain were isolated from a channel catfish clonal B-cell cDNA library. Sequence analysis suggests these cDNAs represent three different MHC class I loci. All cDNAs encoded conserved residues characteristic of the MHC class I alpha chain: namely, those involved in peptide binding, salt bridges, disulfide bond formation, and glycosylation. Southern blot analyses of individual outbred and second-generation gynogenetic fish indicated the existence of both polygenic and polymorphic loci. Northern blot studies demonstrated that catfish B, T, and macrophage cell lines transcribed markedly higher levels of class I alpha and beta2-microglobulin (beta2m) mRNA than fibroblast cell lines. In addition, immunoprecipitation data showed that a 41 000 Mr glycoprotein (presumably class I alpha) was associated with beta2m on the surface of catfish B cells. This latter finding is the first direct evidence for the cell surface association of beta2m with the MHC class I alpha chain on teleost cells and supports the notion that functional MHC class I proteins exist in teleosts.