In order to explore the potential existence of human mast cell growth factors other than stem cell factor (SCF), we have compared SCF to L-cell fibroblast supernatants (LCS) during in vitro mast cell differentiation, using human leukaemic mast cells (HMC-1 cells) which contain a gain-of-function mutated SCF receptor (c-Kit) as model. At baseline, cells exhibited an immature phenotype, with <25% being metachromatic or chloroacetate esterase, tryptase and FcεRIα positive. Intracellular levels of histamine, tryptase, TNF-α and chymase were low, whereas 83% of cells were c-Kit positive. During a 10 day culture with 30% LCS, a significant, time-dependent increase of all mast cell markers, except for chymase and c-Kit, was observed at the protein and for tryptase and FcεRIα also at the mRNA level. Cytoplasmatic granulation and stimulated histamine and leukotriene C4 release were increased as well. In contrast to LCS, rhSCF induced none of these changes in HMC-1 cells. On Sephadex G100 fractionation of LCS, HMC-1 cells increased tryptase activity with fractions between 40 and 60, and below 10 kDa, away from the SCF peak. These data show that HMC-1 cells fail to differentiate in response to SCF and that in addition to SCF, LCS contains other human mast cell growth factors.