Mouse embryonal carcinoma (EC) cells do not express the major H-2 class I transplantation antigens. The latter, however, become detectable upon in vitro differentiation of EC cells. Neither class I H-2 genes nor the gene coding for beta-2 microglobulin (beta 2m) is transcribed in EC cells. We have constructed two hybrid plasmids containing the 5' flanking region of an H-2Kb gene followed by the coding regions of either the herpes simplex virus thymidine kinase (H-2 tk) or the chloramphenicol acetyl transferase (H-2 CAT) genes. Upon transfer into EC cells, the H-2 tk hybrid gene is expressed in F9 tk- cell lines which thus acquire a stable tk+ phenotype. When such transformed clones are induced to differentiate in vitro, tk activity shows a moderate increase, which reflects an increase in transcription of the hybrid gene. In transient transformation experiments, EC cells were found to express the H-2 CAT hybrid gene as well. We conclude that the 2 kilobase pair region of the H-2Kb gene which we used contains an active promoter region, but does not include all the elements required for the correct regulation of the H-2Kb gene.