To investigate the cis-acting DNA elements that are involved in the regulation of class I major histocompatibility complex genes by interferon, several promoter fragments of the H-2Kk gene were linked to the reporter chloramphenicol acetyl transferase (CAT) gene, and the CAT expression was analyzed in stable transfected cell lines. The functional activities of progressive deletions of the 5'-flanking region of the H-2Kk gene linked to the CAT gene have allowed us to define a discrete cis-acting DNA region necessary for interferon-mediated stimulation. Moreover, the H-2Kk gene transcribed by the nonregulated SV40 early promoter was also found to be under interferon regulation. Thus interferon enhancement of the H-2Kk gene expression appears to be mediated by two cis-acting elements, one located in the 5'-flanking region and the other by sequences downstream from the transcription initiation site.