A method was developed for sustained growth of human mammary epithelial cells in monolayer cultures. Epithelial organoids derived from solid breast tissues were grown on the surface of thin (.apprx. 1 mm) collagen gel layers in an enriched growth medium supplemented with hormones, growth factors, fetal calf serum and horse serum. To transfer the cultures, the collagen layers were dislodged and digested with collagenase. The monolayers of cells released into suspension were then dissociated into single cells using trypsin-ethylenediaminetetraacetate. Dissociated single cells were repleted with 75-95% efficiency onto collagen layers or tissue culture plastic surfaces. The dissociated cells could also be cryopreserved and reactivated with > 80% plating efficiency on collagen layers. Normal human mammary epithelial cells grown under these conditions progressed through 12-15 population doublings. The population-doubling time for normal cells on collagen layers was 34. After reaching confluence, cells in some cultures, derived from either normal or malignant tissues, penetrated the gel surface and grew into the collagen. Within the gels, the cells became organized into 3-dimensional tubular structures. The use of collagen layers eliminates a major problem in growth of human mammary epithelial cells in culture, difficulty in efficient dissociation and cell transfer from monolayers.