Pure α-latrotoxin is very inefficient at forming channels/pores in artificial lipid bilayers or in the plasma membrane of non-secretory cells. However, the toxin induces pores efficiently in COS-7 cells transfected with the heptahelical receptor latrophilin or the monotopic receptor neurexin. Signaling-deficient (truncated) mutants of latrophilin and latrophilin-neurexin hybrids also facilitate pore induction, which correlates with toxin binding irrespective of receptor structure. This rules out the involvement of signaling in pore formation. With any receptor, the α-latrotoxin pores are permeable to Ca 2+ and small molecules including fluorescein isothiocyanate and norepinephrine. Bound α-latrotoxin remains on the cell surface without penetrating completely into the cytosol. Higher temperatures facilitate insertion of the toxin into the plasma membrane, where it co-localizes with latrophilin (under all conditions) and with neurexin (in the presence of Ca 2+ ). Interestingly, on subsequent removal of Ca 2+ , α-latrotoxin dissociates from neurexin but remains in the membrane and continues to form pores. These receptor-independent pores are inhibited by anti-α-latrotoxin antibodies. Our results indicate that (i) α-latrotoxin is a pore-forming toxin, (ii) receptors that bind α-latrotoxin facilitate its insertion into the membrane, (iii) the receptors are not physically involved in the pore structure, (iv) α-latrotoxin pores may be independent of the receptors, and (v) pore formation does not require α-latrotoxin interaction with other neuronal proteins.