Uterine expression of lactoferrin (LF) during the preimplantation period and its regulation by the ovarian steroids estradiol (E2) and/or progesterone (P4) in ovariectomized adult mice were examined. Immunoblot detection of LF in uterine cell lysates demonstrated the presence of this protein from days 1-8 of pregnancy [day 1 (D1) = day of vaginal plug]. Immunoprecipitation of 35S pulse-labeled uterine proteins showed that the relative rate of LF synthesis was high on D1, but below the level of detection by D4. Immunolocalization of LF in uterine sections showed intense luminal and glandular epithelial staining on D1 and D2, and progressively decreased staining through D4. Immunoreactive protein was also detected in cells, primarily concentrated in the stroma. The relative number of these cells was greatest on D1 and decreased progressively to a low number by D4. These cells were morphologically similar to neutrophils, which are known to contain LF protein, but little or no LF mRNA. Northern blotting showed that uterine LF mRNA levels were very high on D1 and D2 of pregnancy and decreased to low, but detectable, levels by D4. In situ hybridization to uterine sections showed that LF mRNA was highly abundant only in glandular and luminal epithelial cells, and followed the same pattern as immunolocalization on D1-D4 in epithelial cells. These results document two sources of LF in the preimplantation mouse uterus: neutrophils and epithelial cells. The synthesis of LF in the uterus reflects the abundance of epithelial LF mRNA, which is high on the first 2 days of pregnancy. Neutrophils that contain LF are also abundant in the uterine stroma during this time. E2 and/or P4 regulation of uterine LF was examined. LF mRNA was rapidly induced by E2 in ovariectomized adult mice, and this mRNA was localized exclusively to epithelial cells. P4 had little effect on uterine LF mRNA levels, but antagonized the prolonged induction of this gene by E2. E2 induced the accumulation of immunoreactive LF in uterine epithelial cells and the appearance of numerous immunopositive neutrophils distributed throughout the uterine stroma. P4 also antagonized these effects. Thus, E2 regulates LF gene expression in uterine epithelial cells and causes the recruitment of neutrophils into the uterus. These results suggest that LF may play an important role in early pregnancy and that uterine LF gene expression is regulated by a balance between estrogen and P4.