In the 1950s, Idaho sheepherders observed abnormally high rates of lambs born with craniofacial deformity, referred to as cyclopia. The birth defects were attributed to ingestion of Veratrum californicum. The steroidal alkaloid in V. californicum determined to be the causative agent was cyclopamine. The mechanism of action of cyclopamine is to inhibit the hedgehog (hh) cell signaling pathway. Extraction of dried and ground V. californicum root and rhizome by ethanol yields at least 14 different steroidal alkaloids, of which only eight have been identified and characterized. The six alkaloids that remain to be identified demonstrate hh cell signaling pathway inhibition. Here we present the isolation and characterization of one of the six unknown alkaloids, using high pressure liquid chromatography, nuclear magnetic resonance spectroscopy, mass spectrometry, and circular dichroism spectropolarimetry.