The nicotinamidase activity of cell-free extracts from various mycobacteria was determined qualitatively and quantitatively. Only bovine tubercle bacilli, irrespective of virulence and isoniazid susceptibility, reveal significantly lower nicotinamidase activity than other mycobacteria. The nicotinamidase activity of human, avian, and unclassified mycobacteria is more than 30 times that of bovine bacilli, while that of nonpathogenic acid-fast bacilli is 60 times greater. Thus, bovine tubercle bacilli, both virulent and attenuated, irrespective of isoniazid resistance, can be distinguished from other mycobacteria by determining the nicotinamidase activity of cell-free extracts of the bacteria.