The chemokine receptor CXCR3, which has three known variants (CXCR3-A, CXCR3-B and CXCR3-Alt), has been implicated in the recruitment of mast cells to tissues in many different chronic diseases with its agonists found in elevated levels in many pulmonary diseases. All three variants of CXCR3 were detected in cord blood-derived mast cells at the mRNA level. Using an antibody that is unable to distinguish individual CXCR3 isoforms, we detected a marked down-regulation of intracellular protein during maturation from progenitor cells, with no concomitant changes in the modest surface expression of CXCR3. The known CXCR3 agonists CXCL9, CXCL10 and CXCL11 as well as the reported CXCR3-B agonist CXCL4, were able to induce Akt and ERK1/2 phosphorylation, as well as partial degranulation. Responses to all agonists were inhibited by pre-treatment with selective CXCR3 antagonists and pertussis toxin. Use of novel isoform-selective inhibitors indicates that the p110 isoform of PI3K is required for degranulation and signalling responses to CXCR3 agonists. Unexpectedly, dual (but not individual) isoform inhibition of the class I  and  isoforms substantially inhibited signalling and degranulation responses, indicating a hitherto unrecognised synergy between these isoforms, which provide a conduit for CXCR3 signalling in mast cells.