A previously described gas chromatography-mass spectroscopy (GC-MS) method for the separation and characterization of celestosaminide (celesticetin-related) antibiotics was applied to analyze an unknown celestosaminide mixture from Streptomyces caelestis. Bioactive materials were extracted with methylene chloride at alkaline pH, separated via solvent distribution and silica gel chromatography, and converted to trimethylsilyl (TMS) derivatives for GC-MS analysis. Four new antibiotics (I to IV) were characterized: I and III are celestosamine derivatives (aliphatic esters), while II and IV are lincosamine derivatives (II as an aliphatic ester, IV as an aromatic ester). The production of these antibiotics suggests that the enzymes responsible for ester bond formation have low substrate specificity. Additionally, this work and previous studies indicate the feasibility of directing the production of new celestosaminides by incorporating appropriate acid precursors in the culture medium. The significance of the production of lincosamine-containing antibiotics (II and IV) by S. caelestis in relation to celestosaminide biosynthesis, particularly the sequence of methylation at the C-7 hydroxyl group, is not yet known.