A meiotic recombination hotspot exists within the second intron of the mouse major histocompatibility complex (MHC) gene, Eb. In the present study, a small fragment from the intron which contains two potential transcriptional regulatory elements was cloned into an expression vector and its effect on transcription was tested. This fragment was found to contain tissue-specific transcriptional enhancer activity. An octamer-like sequence and a B motif may contribute to this enhancer activity. Similar regulatory sequences with the same orientation and distance from one another are found in another mouse MHC recombination hotspot.