We have taken the approach of producing somatic cell variants with altered H-2 products to study the structural requirements for cell surface expression of class I histocompatibility molecules. H-2 antigen variants generated by chemical mutagenesis of a cell line expressing the H-2b haplotype were first selected with alloantisera for their loss of H-2Kb expression, and then were analyzed by radioimmunoassay for the appearance of intracellular Kb antigen. For one such variant (69.9.15), whereas the H-2Kb antigen was absent from the cell surface as assayed by antibody-mediated complement-dependent cytotoxicity, an H-2Kb molecule was detected within the cell lysate as confirmed by direct immune precipitation with Kb-specific monoclonal antibodies. The product had an altered antigenic phenotype, since it reacted with only two anti-Kb monoclonal antibodies (Y-3 and EH-144) and not with a third (5F1.2). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis identified the beta 2 microglobulin-associated, intracellular H-2Kb heavy chain to be slightly smaller in Mr than the H-2Kb of the parental cell line. Hybridization analysis revealed the Kb gene from the variant to be without gross alterations, and furthermore, identified a Kb mRNA species that was identical in size to wild-type Kb mRNA. Because complementation was not observed after somatic cell fusion of variant cells with BALB/c splenocytes, it appeared that the alteration in Kb expression was due to a cis-acting defect. In addition, DNA-mediated gene transfer of the wild-type Kb gene into the variant cell line resulted in expression of the Kb antigen on the cell surface, thus confirming that the defect in expression of the mutant Kb product was not due to other factors in the 69.9.15 cell line. Such findings are consistent with the conclusion that stable H-2Kb surface-negative somatic variants can arise due to limited alterations in the Kb gene, resulting in the synthesis of a class I molecule that is expressed only as an intracellular product.