Homeobox-containing genes regulate embryonic developmental programs and are expressed in certain adult tissues and cancers. There has been no report of expression in the breast. We amplified homeobox complementary DNA from mouse mammary gland and found expression of members from each of the four major Hox gene clusters. The regulation of expression of two Hox genes was examined in greater depth. Hoxc-6 transcripts were present in the glands of pubescent and mature mice and decreased during pregnancy. Levels were increased substantially following ovariectomy, indicating possible negative regulation by steroid hormones. Hox expression was studied in mammary adenocarcinomas and in transplant lines of the benign, precancerous tissues from which the cancers arose. Hoxc-6 was expressed at low levels in the precancerous tissue but was not expressed in cancers. In contrast, Hoxa-1 was expressed only in cancers, not in normal gland or in precancerous mammary tissues, suggesting that Hox genes may play a role in a late stage in the stepwise development of mammary malignancies.