The effect of embryo fibroblasts on the growth of erythroid colony-forming cells in vitro (CFU-e) from mouse bone marrow was investigated. First, the maintenance of CFU-e number in suspension culture was assayed. CFU-e recovered from suspension culture fell rapidly to values below 30% of the initial number. When erythropoietin (EP) was added, the initial decline during the first day was followed by a rise to 80%. In cultures supplemented with irradiated fibroblasts, the number of CFU-e did not show an abortive fall, but there was a slight increase during 3 days of culturing. The influence of fibroblasts on the colony-forming ability of CFU-e was studied in a semisolid culture system composed of an agar underlayer and a methylcellulose overlayer. The number of erythroid colonies scored after 5 days of culture in the presence of different levels of EP was proportional to the number of added fibroblasts and the colony size (depending on the number of fibroblasts) increased to macroscopic dimensions. Fibroblasts alone, without EP, induced colony formation by CFU-e if added in concentrations of 1 X 10(5) or higher. EP was not detectable in medium conditioned by the fibroblasts. These data indicate that fibroblasts may stimulate erythroid colony formation (in the absence of EP) and enhance the colony-forming ability of CFU-e in the presence of EP. From these results, it is suggested that fibroblasts exert proliferation activating effects on CFU-e target cells.