Adenine nucleotides in Escherichia coli, Bacillus cereus, Klebsiella pneumoniae, Staphylococcus aureus, and Pseudonzonas aeruginosa were extracted using 10 different methods. Extracts were assayed for adenosine 5'-triphosphate (ATP), and adenosine 5'-diphosphate (ADP), and adenosine 5'-monophosphate (AMP) by the firefly method using an improved procedure. Analytical interference by bacterial enzymes not inactivated during the extraction was found to be a major problem. However, these enzymes were inactivated to a considerable extent by the inclusion of ethylene-diaminetetraacetate in the extraction reagent. The 10 extraction methods were compared with respect to yield of adenine nucleotides, interference with the enzymic assay, reproducibility of the method, and stability of the extracts. Results indicated that extraction with trichloroacetic acid was the method most closely reflecting actual levels of ATP, ADP, and AMP in intact bacterial cells. However, for the extraction of ATP in some bacterial strains several other methods may be used and may be advantageous from a practical point of view.