The marrow stromal fibroblast (MSF) population has been shown to include precursor cells for at least five types of connective tissue: bone, cartilage, adipose tissue, fibrous tissue, and hematopoiesis-supporting reticular stroma. In this study, growth requirements for MSF colony formation were studied in vitro. In order to exclude the influence of nonadherent cells, after a period of initial adhesion of bone marrow cells in serum-containing medium nonadherent cells were removed. Further cultivation was carried out in either serum-containing or serum-free conditions, with or without feeder cells (irradiated bone marrow cells). This approach revealed differences between animal species in initial MSF growth requirements. In serum-containing conditions, mouse MSF precursor cells (colony-forming units-fibroblast, CFU-Fs) were shown to be feeder cell dependent: MSF colonies were formed only in the presence of feeder cells. Guinea pig CFU-Fs were partially feeder cell dependent, whereas human CFU-Fs were feeder cell independent. In serum-free conditions, CFU-Fs of all three species were feeder cell dependent. The difference between the growth requirements for mouse and human MSFs was not caused by serum origin or concentration, feeder cell origin, or differences in the preparation of marrow cell suspensions.