BACKGROUND The identification of potential breast cancer stem cells is of importance as the characteristics of stem cells suggest that they are resistant to conventional forms of therapy. Several techniques have been proposed to isolate or enrich for tumorigenic breast cancer stem cells, including (a) culture of cells in non-adherent non-differentiating conditions to form mammospheres and (b) sorting of the cells by their surface phenotype (expression of CD24 and CD44). METHODS We have cultured metastatic cells found in pleural effusions from breast cancer patients in non-adherent conditions without serum to form mammospheres. Dissociated cells from these mammospheres were used to determine the tumorigenicity of these cultures. Expression of CD24 and CD44 on uncultured cells and mammospheres derived from the pleural effusions was documented. RESULTS We found that the majority (20/27) of the pleural effusions tested contained cells capable of forming mammospheres of varying sizes that could be passaged. After dissociation and plating with serum onto adherent dishes, the cells can differentiate, as determined by the increased expression of cytokeratins and MUC1. Analysis of surface expression of CD24 and CD44 on uncultured cells from 21 of the samples showed that the cells from some samples separated into two populations, but some did not. The proportion of cells that could be considered CD44+/CD24low/- was highly variable and did not appear to correlate with the ability to form the larger mammospheres. Of eight pleural effusion mammospheres tested in severe combined immunodeficiency disease (SCID) mice, four were found to induce tumours when only 5,000 or fewer cells were injected, whereas the same number of uncultured cells did not form tumours. The ability to induce tumours appeared to correlate with the ability to produce the larger mammospheres. Uncultured cells from a highly tumorigenic sample (PE14) were uniformly negative for surface expression of both CD24 and CD44. CONCLUSIONS This paper shows, for the first time, that mammosphere culture of pleural effusions enriches for cells capable of inducing tumours in SCID mice. The data suggest that mammosphere culture of these metastatic cells could provide a highly appropriate model for studying the sensitivity of the tumorigenic 'stem' cells to therapeutic agents and for further characterisation of the tumour-inducing subpopulation of breast cancer cells.