As a suitable model to study the growth behavior and therapeutic response of drug-resistant and -sensitive cells in three-dimensional coculture we have established multicellular spheroids generated from both cisplatin-sensitive and -resistant cells of a murine fibrosarcoma cell line. A drug resistant clone was derived from the parent cisplatin-sensitive cells by intermittent drug exposure in vitro. As a prerequisite for analysis of differential growth and treatment response of spheroid subpopulations, two efficient methods to discriminate between the two morphologically indistinguishable subpopulations in mixed spheroids were established. In the cisplatin-resistant cell line chosen for the present study, resistance is mainly due to an increased cellular metallothionein content and is therefore associated with increased resistance to CdCl2. Exposure of colonies to high concentrations of CdCl2 thus allowed selective elimination of sensitive colonies. Permanent labeling of either resistant or sensitive cells was achieved by introduction of the Escherichia coli beta-galactosidase marker gene with a retroviral vector system. The transformation of an uncolored galactose derivative by this enzyme into an indigo stain allowed detection of cells carrying and expressing the marker gene. The marker gene and CdCl2 method led to identical results when used simultaneously to distinguish quantitatively between resistant and sensitive colonies grown from plated cells of untreated or irradiated mixed spheroids. The retroviral labeling method was also used successfully in the study of intact spheroids, showing that in 1:1 mixed spheroids, cisplatin-sensitive parent cells accumulate in the spheroid periphery, outgrowing resistant cells and displacing them into the metabolically restricted spheroid center. Only when sensitive and resistant cells are initially mixed at a ratio of 1:9 are the resulting spheroids composed of equal proportions of the 2 cell types throughout 10-20 days after spheroid initiation.