The antiallergic drug oxatomide and analogs inhibit mediator release from a rat basophilic leukemia (RBL-2H3) cell line, which is frequently used as a mast cell model. By investigating a series of derivatives of oxatomide with different inhibiting activities on exocytosis, we aimed to evaluate the role of their effects on the early steps of the signal transduction cascade in the inhibition of exocytosis. The active compounds induced hyperphosphorylation of tyrosine residues both in stimulated as well as in resting cells. Furthermore, some elevation of the inositol 1,4,5-trisphosphate (IP3) formation upon antigen activation was observed for the active derivatives. Ca2+ fluxes were also studied. The inhibition of the antigen-induced 45Ca2+ influx correlated with the effects of the drugs on exocytosis. Furthermore, the inhibitory activity on antigen- and thapsigargin-mediated exocytosis correlated well. Adherence of the cells to fibronectin, stimulating cellular integrin receptors, was synergistic to antigen activation of the RBL cells. However, oxatomide did lack any effect on integrin-mediated processes, as the IC50 value for exocytosis was identical for fibronectin-adhered cells and standard cultured cells. We conclude that oxatomide and its analogs inhibit exocytosis, mainly by inhibiting Ca2+ influx over store-operated Ca2+ (SOC) channels. The drugs have a direct effect on the store-operated Ca2+ channels or affect the direct regulation of these channels.