Interleukin-3 (IL-3) has been shown to have significant effects on haemopoiesis in vitro, but early investigations of normal human long-term bone marrow cultures (LTBMC) have failed to demonstrate IL-3 production by stromal cells, either by Northern blotting for mRNA, or assaying for bioactivity in culture supernatants. One recent report, using reverse transcription-polymerase chain reaction (RT-PCR), demonstrated IL-3 expression in only one of eight cultures. We have developed a sensitive bioassay for the detection of IL-3 production from normal stroma in LTBMC. LTBMC were grown until confluent, irradiated, and stroma harvested by trypsinization to yield single-cell suspensions. These cells were then cocultured with target bone marrow mononuclear cells (BMMC), or CD34+ cells in clonogenic assays, either in the presence or absence of anti-IL-3 neutralizing antibodies. We have demonstrated IL-3 production in 32/34 cases. In addition, by separating stroma from target cells using cell culture inserts, we have shown that direct stroma:stem cell contact is not necessary for colony growth, suggesting that IL-3 diffuses into the supernatant. However, when supernatants from LTBMC were assayed by enzyme-linked immunoassay (ELISA), no IL-3 was detected. This suggests that IL-3 is probably produced at low levels and has a short-range interaction. Stroma production of IL-3 was confirmed by the detection by RT-PCR of IL-3 mRNA in 3/3 cases. The simultaneous detection of CD2 mRNA demonstrated that T cells are part of the bone marrow stroma. It is therefore possible and probably likely that these cells are the source of IL-3.