Intramammary infections (IMI) are prevalent in nonlactating dairy cattle and are known to impact mammary structure and negatively affect the amount of mammary epithelium in the gland. It is currently unknown what chief mechanisms involved with mammary growth are affected by the presence of an IMI. This study’s objective was to characterize the expression of estradiol receptor α (ESR1) and progesterone receptor (PGR) in mammary glands that were stimulated to grow and develop in the presence or absence of an IMI. Mammary growth was stimulated in 18 nonpregnant nonlactating dairy cows, using estradiol and progesterone injections, and 2 culture negative quarters of each cow were subsequently infused with either saline (n = 18) or Staphylococcus aureus (n = 18). Mammary parenchyma tissues were collected 5 d (n = 9) or 10 d (n = 9) post-challenge and examined using immunofluorescence microscopy. Data were analyzed using a mixed model with treatment and euthanasia day as fixed effects and cow nested within euthanasia day as a random effect. The number of ESR1-positive nuclei, observed across 8 random mammary parenchyma fields of view for each quarter, tended to be greater in saline quarters than challenge quarters (201 vs 163 ± 44 nuclei; P = 0.1) and were lower in tissues collected 5 d post-challenge than 10 d (130 vs. 234 ± 59 nuclei; P = 0.04). Saline quarters contained a greater number of PGR positive nuclei than challenged quarters (520 vs 440 ± 45 nuclei; P = 0.024) but, positive PGR nuclei counts were not influenced by tissue collection day (P = 0.78). The area and intensity of nuclear ESR1 and PGR staining was differentially affected by the effects of quarter treatment and day of tissue collection. These results indicate that IMI in nonlactating glands, that were stimulated to grow, affects the number of nuclei expressing ESR1 and PGR and alters expression characteristics of these receptors in the nucleus. Alterations in estradiol and progesterone signaling in mammary parenchyma would likely affect the signaling pathways of these key hormones involved with mammary growth and development.