Identification and initial characterization of an apparently large, membrane-associated multifunctional enzyme, kinamycin acetyltransferase I (KAT I), is described. KAT I activity was enriched 29-fold over the level in cell-free extracts of Streptomyces murayamaensis. Two acetyltransferase activities catalyzing acetyl coenzyme A dependent conversion of kinamycin F and E to kinamycin E and D, respectively, were inseparable in the course of the partial purification. Partial purification involved separation of KAT I from cytosolic proteins by differential ultracentrifugation, solubilization with 0.5% CHAPS zwitterionic detergent followed by ultracentrifugation, and Sephacryl S400 gel filtration chromatography of the resulting supernatant.