Cells were obtained from the mammary glands of sheep and cows by collagenase–hyaluronidase digestion. Characterization of cells as epithelial was by reaction with a monoclonal antibody to cytokeratin. A subpopulation of spindle-shaped or stellate cells reacted with a monoclonal antibody to desmin and may be related to myoepithelial cells. The development is described of a simple serum-free culture system for these cells on gels of rat tail (type 1) collagen. A commercial medium (M199) was used, buffered with Hepes and with bovine serum albumin as the sole protein supplement, plus fibronectin for the first 18 h only as an attachment factor. The cell cultures showed stimulated DNA synthesis in response to mitogens on attached gels and also responded as floating cultures to lactogenic hormones with production of α-lactalbumin.