A method developed for the initiation and maintenance in primary culture of human normal mammary epithelial cells was adopted for the growth of epithelial cells from 45 primary human breast tumors. The cells were grown on a naturally produced extracellular matrix (ECM) or on regular tissue culture plastic in a serum-free medium containing growth supplements and high-density lipoprotein (HDL). Successful enzymatic dissociation of the tumor biopsy into organoid structures and cell aggregates was crucial for subsequent cell attachment and growth. Fifty-five percent of the biopsy specimens were successfully dissociated and 87% of these gave rise to actively dividing epithelial cells forming monolayer cultures. In contrast, only 21% of the biopsies which were not optimally dissociated yielded growing cultures. Variations in sample size, duration of enzymatic digestion, and tumor composition affected the outcome of tumor dissociation. Omission of serum from the culture medium prevented the growth of fibroblasts, while plating on ECM greatly improved and in some cases was essential for cell attachment and subsequent outgrowth. The epithelial nature of the cells was verified by their cuboidal and closely apposed morphology and positive staining with antikeratin antibodies. The growth and subculture requirements and the expression of the B38.1 tumor marker were compared in human mammary epithelial cells derived from solid tumors, pleural effusion and normal breast tissue.