The synthesis of a series of pyridothienopyrimidines and their evaluation as inhibitors or inducers of the release of histamine from rat mast cells is reported. The activity was measured after immunological stimulation with ovoalbumin and chemical stimulation with polymer 48/80 and the drugs adryamicin and vinorelbine. The experiments were carried out with and without preincubation of the stimulus with the cells before addition of the drug. Several pyridothienopyrimidines show inhibitory IC50 values in the range 2–25 μM, indicating they are up to 100 times more potent than cromoglycate (DSCG) and 10 times greater than Ketotifen. Compound 9l is a potent inhibitor in all the conditions tested and shows IC50=9–25 μM. Pyridothienopyrimidines 4l and 9e are very strong inducers of histamine release in the immunological (4l, 170–230%) and chemical (9e, 100–150%) assays, respectively. Compounds 4l and 9i are cytotoxic in vitro (IC50=0.1–0.2 μg/mL) against P-388, A-549, HT-29, and MEL-28 tumor cell lines.