Trop-2/EGP-1/GA733-1 is a recently identified cell surface glycoprotein highly expressed by human carcinomas. The cytoplasmic tail of Trop-2 possesses potential serine and tyrosine phosphorylation sites and a phosphatidyl-inositol binding consensus sequence. Thus, we investigated whether Trop-2 might be a functional signaling molecule. Using the fluorescent probe Fura-2, we assayed the cytoplasmic calcium levels in human cancer cells stimulated with anti-Trop-2 or control antibodies. Three anti-Trop-2 MAbs, Rs7-7G11, MOv16 and 162-46.2 specifically induced a transient intracellular calcium level increment in up to 40% of the experiments performed. Polyclonal antisera recognizing recombinant Trop-2 molecules possessed a much lower stimulation efficiency. The average latency of antibody-induced Ca 2Ψ rise for OvCa-432 cells was 64 Ψ 26 sec. Internal Ca 2Ψ concentrations reached peaks of 190 Ψ 24 nM vs. basal levels of 61 Ψ 4 nM and returned to baseline within 193 Ψ 37 sec. Similar values were obtained in MCF-7 cells. For comparison, stimulation of P2-purinergic receptors on MCF-7 and OvCa-432 cells induced a Ca 2Ψ rise in most cases, leading to average internal Ca 2Ψ concentrations of 297 Ψ 41 and 391 Ψ 39 nM, respectively. Our findings show that Trop-2 transduces an intracellular calcium signal, are consistent with the hypothesis that it acts as a cell surface receptor and support a search for a physiological ligand.