To date, two classes of mouse major urinary protein (MUP)-encoding genes have been described, the expressed genes and the intervening-sequence-containing pseudogenes. The data presented in this paper define a third class, the silent Mup genes, which are potentially functional but appear not to be expressed under normal circumstances. We describe a MUP subfamily (Mup-1.5) containing two genes, Mup-1.5a and Mup-1.5b, that are nearly identical, differing at only three positions (greater than 99.9% identity) over the entire 4-kilobase (kb) transcription unit and approximately 1 kb of flanking DNA. The similarity between these two genes extends over greater than 35 kb. Using specific oligonucleotides, we have shown that the 5a gene is expressed in BALB/cByJ mice, primarily in the submaxillary gland, whereas the 5b gene is not expressed. However, we found that when a 9.4-kb DNA fragment containing the Mup-1.5b gene was introduced into the mouse germ line, mice in two of the four transgenic lines expressed this gene at a high level and with the tissue-specificity characteristic of the Mup-1.5a gene. These results suggest that the inactivity of the endogenous Mup-1.5b gene is due not to a lack of functional positive regulatory elements, but to long-range, inhibitory position effects.