It has been reported that rare colonies of mast cells may be identified when human bone marrow cells are cultured in agar. Based on this observation, we attempted to culture mast cells from human bone marrow using a modified agar interphase culture. Metachromatically staining cells appearing in culture peaked in number by 2-3 weeks and consisted of basophil-like cells, mast-like cells and larger, granulated cells superficially resembling mast cells. The large cells, which constituted the majority of metachromatic cells, were berberine sulphate-positive but negative for histamine, chloroacetate esterase, and mast cell tryptase. These larger granulated cells were subsequently identified as macrophages by FACS analysis with Leu M3 and by electron microscopy. Berberine sulphate-staining of macrophages was not due to the de novo synthesis of heparin, as shown by analysis of radiolabelled proteoglycans from cultured cells. The macrophage metachromasia was eliminated with the use of agarose. The metachromatic and berberine sulphate-staining of cultured human macrophages was therefore considered to be due to phagocytosed agar. Recognition of this phenomenon will aid in the proper identification of human mast cells and basophils in cultures where agar or similar substances are present, and demonstrates that berberine sulphate-staining is not specific for heparin.