Abstract The structure of acumycin, an antibiotic isolated from Streptomyces griseoflavus, was determined from spectroscopic and X-ray studies. Acumycin was found to be a 16-member ring macrolide with two sugars attached. One of the sugars had earlier been shown to be D-mycaminose. From the known absolute configuration of D-mycaminose we have been able to determine the absolute stereochemistry of acumycin. The stereochemistry in acumycin is compared with the predictions based on a biogenetic model proposed by Celmer. Acumycin because of its large number of chiral centers provided the most demanding test of the model to date and the model was found to be consistent with the experimental results from acumycin in every detail but one. These results suggest a refinement of Celmer's model for 16-member ring macrolides.