To accurately delineate the DNA sequence elements involved in the transcriptional regulation of the murine MHC class II gene E alpha, we have constructed a set of "linker-scanning" mutants of the E alpha promoter region. These were made by the gapped heteroduplex technique and result in a set of 10-bp replacement mutations, covering the -206 to -6 stretch of the promoter. The effect of these mutations on transcriptional activity was evaluated in several systems, either by transfection into cultured cells or by in vitro transcription. The data points to the now classical X and Y boxes as the most important control elements, either for constitutive expression in B lymphoma cells, or for IFN-gamma-inducible expression in macrophages. Motifs upstream of the X box also play a role, but are somewhat less critical. Overall, we find no marked difference between regulatory strategies in B lymphomas or activated macrophages.