alpha-Latrotoxin (alpha-LTx), a vertebrate neurotoxin isolated from Black Widow Spider venom, causes massive spontaneous neurotransmitter release. The molecular mechanism(s) by which the toxin exerts its effect is largely unknown. Here we report identification and purification of a novel membrane receptor with high affinity for alpha-LTx. Unlike neurexin Ia, a previously described high affinity alpha-LTx receptor, this novel protein binds alpha-LTx independently of Ca2+ presence and therefore may be a mediator of the calcium-independent stimulation of neurotransmitter release by alpha-latrotoxin. The major protein component of calcium-independent alpha-LTx receptors is a novel M(r) 120,000 protein which does not belong to the neurexin family. Among several tissues tested, the M(r) 120,000 protein was found only in brain.