We directly compared the mammary stem cell (MaSC) enrichment of two well-known MaSC isolation protocols; CD24+CD49fhi and CD24+ Sca-1neg. This revealed that the CD24+CD49fhi population is 2.5-fold more enriched for stem cell activity. As the CD49fhi population contains a Sca-1+ population, we postulated that exclusion of Sca-1 positive cells accounts for reduced MaSC enrichment within the CD24+Sca-1neg population. To determine if this was the case we assessed various MaSC characteristics including abundance, level of quiescence, in-vivo repopulating activity and gene expression. Assessment of the repopulating potential of the CD24+CD49fhiSca-1- and CD24+CD49fhiSca-1+ population revealed Sca-1 positivity in addition to CD24+CD49fhi does not increase MaSC enrichment. Cell cycle analysis showed that at 3 weeks the entire MaSC compartment is quiescent, but at 6 weeks of age the Sca-1+ subset is actively cycling whilst the Sca-1- subset remains relatively quiescent. The CD24+CD49fhiSca-1+ were highest in pre-pubescent mice (3 weeks) and then upon the onset of puberty reduced in number such that they were significantly decreased compared to CD24+CD49fhiSca-1- at 9 and 25 weeks of age. The CD24+ CD49fhi and Sca-1neg subset of the former, were both increased with age. Gene expression analysis showed markers of Stem and luminal cells, as well as a 20- and 5- fold up-regulation of estrogen receptor (ER) α in the Sca-1+ subset at 3 and 6 weeks respectively. ER protein expression was then confirmed by immunofluorescence staining. This work shows for the first time that the MaSC compartment of the mammary gland contains an ERα positive subset that decreases with age.