3-Amino-3-deoxy-alpha-D-glucopyranosyl-alpha-D-glucopyranoside (alpha, alpha--3-trehalosamine) was isolated from a culture of Nocardiopsis trehalosei sp. nov. (NRRL 12026). The structure was determined using a combination of spectroscopic techniques on derivatives of the component sugars, especially gas chromatography-mass spectrometry. The compound exhibited antibiotic activity against Gram-positive organisms at levels similar to what was found for the 2- and 4-trehalosamines.