Elevated aldehyde dehydrogenase (ALDH) expression/activity has been identified as an important biomarker of primitive cells in various normal and malignant human tissues. Here we examined the level and type of ALDH expression and activity in different subsets of phenotypically and functionally defined normal human mammary cells. We find that the most primitive human mammary stem and progenitor cell types with bilineage differentiation potential show low ALDH activity but undergo a marked, selective, and transient upregulation of ALDH activity at the point of commitment to the luminal lineage. This mirrors a corresponding change in transcripts and protein levels of ALDH1A3, an enzyme involved in retinoic acid synthesis and the most highly expressed ALDH gene in normal human mammary tissue. In contrast, ALDH1A1 is expressed at low levels in all mammary epithelial cells. These findings raise interesting questions about the reported association of ALDH activity with breast cancer stem cells and breast cancer prognosis. STEM CELLS 2012; 30:344–348.