Culture of human mast cells (MC) in vitro has only been possible to date in the presence of 3T3 fibroblasts. The aim of the present study was to maintain freshly isolated human MC in culture without addition of feeder cells and to study their functional properties. We isolated cell suspensions containing 1 to 11% MC from human intestinal tissue and cultured them in standard medium. MC survived in culture for about 2 wk without cytokine supplementation and for several months with supplementation of medium with stem cell factor (SCF). SCF selectively supported MC survival, whereas the number of contaminating cells declined rapidly during culture. Most interestingly, we found that histamine and leukotriene release induced by IgE receptor cross-linking was substantially enhanced in cultured MC compared with that in MC stimulated directly after cell isolation. Cultured MC, but not freshly isolated MC, released mediators in response to SCF in a concentration-dependent fashion provided that the cells were cultured in SCF-free medium. These findings demonstrate that human MC isolated from intestinal tissue can be maintained in culture in vitro for several weeks. After culture they have different functional properties, which might resemble more closely the functional status of human intestinal MC in vivo than that of freshly isolated cells.