A three-dimensional bone-marrow culture system was utilized for assessing the toxicity of various chemotherapeutic agents. This model, which exhibits multilineage haematopoiesis and promotes the growth of progenitor cells for extended periods, was treated with various concentrations of beta-d cytosine arabinofuranoside (Ara-C), cyclophosphamide (CP), methotrexate (MTX) or 5-fluorouracil (5-FU). The effects of these agents on the phenotypes of adherent zone cells was ascertained by labelling with monoclonal antibodies against rat leucocytes and evaluating by flow cytometry. Adherent zones also were assayed for content of colony-forming unit culture (CFU-C) and cellular viability after drug exposure was measured using the neutral red (NR) assay. The results indicated that Ara-C, CP, MTX and 5-FU treatment caused dose-dependent decreases in the CFU-C concentration of adherent zones in cultures of various ages. These agents also altered the phenotypic distribution of adherent zone cells and displayed differential lineage specificities. A dose-related decrease in viability also was observed with the NR assay, albeit at higher drug doses than those which induced measurable CFU-C and phenotypic alterations. These three-dimensional cultures may prove to be ideal substrates for toxicity testing as they contain all of the cell types present in vivo, are physiological with respect to their growth patterns, are easily manipulable, and can be maintained for extended periods.