In experimental animal models the susceptibility of the mammary gland to neoplastic transformation is related to its degree of development and proliferative activity; this observation led us to determine whether the human breast epithelium also exhibits development-related differences, and whether these differences could be detected in an in vitro system. Normal breast tissue obtained from reduction mammoplasties of 9 patients ranging in age from 18 to 56 years were characterized in both whole mount preparations and organoids obtained after collagenase-hyaluronidase digestion by their degree of development based upon the types of lobules present. Lobules were classified into type 1 (Lob 1), composed of approximately 11 alveolar buds, the less developed; lobules type 2 (Lob 2), of moderate development, composed of approximately 47 ductules each, and lobules type 3 (Lob 3), composed of 80 ductules each, represented the highest level of development. Epithelial organoids obtained after digestion were plated in DMEM:F12 medium supplemented with hydrocortisone, cholera toxin, insulin and 5% horse serum with a calcium concentration of 1.05 mM Ca++. Following attachment, the medium was replaced by medium containing 0.040 mM Ca++. The percentage of attachment of organoids to the flask was greater in cells from Lob 1 (89-99%) and Lob 1+2 (79-100%) than in cells from Lob 3, which had a 53-67% attachment. The total yield of cells after 7 weeks in culture was also greater in cells derived from Lob 1 and Lob 1+2 than in cells from Lob 3. The total yield of cells obtained from primary cultures was not related to the number of organoids plated, but to the degree of development of the gland. The DNA-labeling index (DNA-LI) in intact breast tissue correlated with that in primary cultures; it was greater in Lob 1 and Lob 1+2 than in Lob 3. By flow cytometry, the highest percentage of cells in S-phase was seen in cells with the highest DNA-LI. We concluded that the growth characteristics of mammary epithelial cells in vitro in a low Ca++ medium is modulated by the degree of development and differentiation of the gland.