Brasilinolide A (1) is a polyhydroxy macrolide possessing potent immunosuppressive activity isolated from a pathogenic actinomycete Nocardia brasiliensis IFM 0406. The gross structure and partial relative stereochemistry of 1 have been elucidated by 2D NMR data, whereas its absolute stereochemistry remains undefined. Further investigation resulted in isolation of a new related macrolide, brasilinolide C (2), and assignments of the absolute configurations of 1 and 2 on the basis of spectroscopic data and chemical means as follows. Solvolysis of 2 afforded a macrocyclic aglycone (3) and L-2-deoxyfucopyranose (4). Oxidative degradation of the aglycone (3) gave three segments, C1-C15 (7), C17-C27 (8), and C28-C37 (9). On the other hand, oxidative degradation of 2 yielded two segments, C1-C15 (14) and C17-C37 (15). The relative stereochemistry was elucidated mainly by NMR data including NOESY correlations for the acetonide derivatives (10-14 and 15) or the oxidative product (16). Absolute configurations were assigned on the basis of modified Mosher's method applied to C5, C9, C13, C19, and C31. Finally, the absolute configurations at 26 chiral centers in 2 were elucidated. Similarly, the absolute configurations of brasilinolide A (1) were also assigned as the same as 2.