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A battery of morphological, physiological, and biochemical tests, including paper chromatographic analysis of whole cell hydrolysates, was used to study 177 cultures of aerobic actinomycetes. One hundred thirty-five of the 177 were submitted as diagnostic laboratory specimens; of these, 129 were identified as belonging to 1 of 10 genus or species groups. The tests and procedures used were found to be relatively easy to perform and interpret. On the basis of the results, a flow chart was devised to permit the step by step identification of unknown clinical isolates of these organisms.