A technique is reported here for the quantitative extraction of live cells from the lung interstitium; it involves the incubation of slices of perfused lung in a mixture containing optimal concentrations of collagenase, DNAse, and fetal calf serum, followed by the simultaneous recovery and fractionation of cells released from the tissue matrix on a six-step discontinuous percoll gradient. Yields in the order of 10(8) viable cells per gram of lung were routinely achieved with tissues from rat, mouse and guinea-pig. Preliminary characterization of these cells has been performed in the rat by histological techniques (Giemsa staining, transmission electron microscopy), cytochemistry (acid phosphatase, esterase, peroxidase), by the capacity to bind monoclonal antibodies directed at lymphocyte surface markers, and by a range of functional tests. The cells comprised, on average, 32% macrophages, 44% lymphocytes (T and B cells and large granular lymphocytes), with small numbers of eosinophils, mast cells and epithelial cells. Transmission electron microscopy revealed minimal ultrastructural damage to extracted cells, with such functions as phagocytosis, FcR activity, mitogen responsiveness, antigen presentation, and NK-cell activity, being readily demonstrable. In addition, these activities segregated into defined areas of the six-step density gradient.