ABSTRACT Streptomyces ambofaciens is prone to genetic instability involving genomic rearrangements at the extremities of the chromosomal DNA. An amplified DNA sequence (ADS205), including an open reading frame ( orfPS ), is responsible for the reversible loss of spiramycin production in the mutant strain NSA205 (ADS205 ⁺ Spi ⁻ ). The product of orfPS is homologous to polyketide synthase systems (PKSs) involved in the biosynthesis of erythromycin and rapamycin and is overexpressed in strain NSA205 compared with the parental strain RP181110. As PKSs and fatty acid synthase systems have the same precursors, we tested the possibility that overexpression of orfPS also affects lipid metabolism in strain NSA205. This report focuses on comparative analysis of lipids in strain RP181110, the mutant strain NSA205, and a derivative, NSA228 (ADS205 ⁻ Spi ⁺ ). NSA205 showed a dramatically depressed lipid content consisting predominantly of phospholipids and triacylglycerols. This lipid content was globally restored in strain NSA228, which had lost ADS205. Furthermore, strains RP181110 and NSA205 presented similar phospholipid and triacylglycerol compositions. No abnormal fatty acids were detected in NSA205.