Acyl carrier protein (ACP) activity was determined by the malonyl-coenzyme A–CO 2 exchange reaction. It was highest in Acholeplasma laidlawii , lower in A. granularum , and lowest in A. axanthum . The sterol-requiring Mycoplasma species examined showed little or negligible ACP activity. A. laidlawii was capable of utilizing pantetheine or coenzyme A but not β-alanine as precursor for ACP synthesis. Its ACP could thus be labeled by growing the organisms with radioactive coenzyme A. The ACP of A. laidlawii appears to be a soluble cytoplasmic protein, which could be purified about 40-fold by treatment of the cytoplasmic fluid with streptomycin sulfate and chromatography of the supernatant fluid on a Biogel P-10 column. Its molecular weight, determined by polyacrylamide gel electrophoresis, is low (about 10,900) resembling that of Escherichia coli , but it is much more sensitive to heat.