Abstract A cell separation method was developed for studies of the growth kinetics of rat mammary epithelial cell (RMEC) subpopulations in grafts and in culture in vitro . By flow cytometry of RMEC stained with fluorescein isothiocyanate-peanut agglutinin and phycoerythrin-anti-Thy-1.1 monoclonal antibody, we could distinguish four cell subpopulations from primary cultures of 7- to 8-week-old F344 female rat mammary glands: both negative (B-), PNA+, Thy-1.1+, and both positive (B+). We studied the growth patterns of these subpopulations in vitro for 1 to 14 days in complete hormone medium (CHM) containing 10% fetal bovine serum and prolactin, 17β-estradiol, cortisol, progesterone, and insulin. The fractions of PNA+ and B- cells steadily decreased with time in culture. The fraction of Thy-1.1+ cells steadily increased with time in culture. There were small numbers of B+ cells. We grafted RMEC in hyperprolactinemic recipient rats. The mean numbers of transplanted cells required to produce at least one alveolar unit in 50% of the graft sites (AD 50 values) are inversely related to the clonegenie fractions. AD 50s of RMEC are as follows: unsorted mammary cells cultured in CHM for 1 to 4 days, ∼220; unsorted cells cultured in CHM for 7 days, ∼550; sorted PNA+ RMEC from outgrowths of 3-day cultures, ∼82; B+, ∼350; B-, ∼545; Thy-1.1+, ∼9372. We conclude that the PNA+ cell subpopulation includes most of the clonegenic cells. Thy-1.1+ cells appear to he terminally differentiated; they are likely to be myoepithelial cells.