<jats:title>Abstract</jats:title><jats:p>Two new and a known <jats:italic>N</jats:italic>‐(<jats:italic>O</jats:italic>‐carbamoylglucopyranosyl)ansamitocins were isolated from <jats:italic>Actinosynnema pretiosum</jats:italic> ssp. <jats:italic>auranticum</jats:italic> ATCC 31565. The known <jats:italic>N</jats:italic>‐(4‐<jats:italic>O‐</jats:italic>carbamoyl‐<jats:italic>β</jats:italic>‐<jats:sc>D</jats:sc>‐glucopyranosyl)‐<jats:italic>N</jats:italic>‐demethylansamitocin P 2 (=ACGP‐2; <jats:bold>1</jats:bold>) was assigned according to 1D‐ and 2D‐NMR data, and the two new compounds were identified as <jats:italic>N</jats:italic>‐(6‐<jats:italic>O‐</jats:italic>carbamoyl‐<jats:italic>β</jats:italic>‐<jats:sc>D</jats:sc>‐glucopyranosyl)‐<jats:italic>N</jats:italic>‐demethylansamitocin P 2 (=ACGP‐2′; <jats:bold>2</jats:bold>) and <jats:italic>N</jats:italic>‐(4‐<jats:italic>O‐</jats:italic>carbamoyl‐<jats:italic>β</jats:italic>‐<jats:sc>D</jats:sc>‐glucopyranosyl)‐<jats:italic>N</jats:italic>‐demethylansamitocin P 1 (=ACGP‐1; <jats:bold>3</jats:bold>) on the basis of spectroscopic data interpretation including 2D‐NMR and tandem MS analysis.