HBL100 is a human breast epithelial cell line which has been immortalized by the defective genome of SV40 but which is not tumorigenic in nude mice. Growth experiments in defined serum-free and protein-free media revealed its ability to grow without exogenous supply of serum growth factors. Addition of heparin to the serum-free medium increased its growth rate, suggesting the release of heparin-binding autocrine growth factor(s) in the culture medium. HBL100-conditioned medium was mitogenic for HEMG (human embryonic mammary gland) and CCL39 fibroblasts. Western blot analysis of growth factors revealed the presence of the M(r) 18,000, 22,000 and 24,000 forms of basic fibroblast growth factor (bFGF or FGF2) both in HBL100 cell extracts partially purified on heparin-Sepharose beads and in concentrated conditioned medium. Northern blot analysis of HBL100 RNA demonstrated the synthesis of 7-, 3.7-, and 1.8-kilobase (kb) FGF2-specific mRNAs by these cells. Anti-FGF2 neutralizing IgG inhibited HBL100 growth in serum-free medium in a dose-dependent manner. Affinity cross-linking of 125I-FGF2 to the HBL100 cell surface revealed an FGF receptor of 130 kDa. Taken together, these results suggest that FGF2 may be implicated as an autocrine growth factor in the early events leading to neoplastic transformation of human breast epithelial cells.