Acyl-CoA carboxylase activity in four pathogenic mycobacteria and Mycobacterium smegmatis was shown with both acetyl-CoA and propionyl-CoA substrates. Only very low activity was detected in mycobacteria grown in host tissues or on egg-based media rich in lecithin and avidin. This appeared to be a result of severe depression of activity, as strains which could be grown both in host tissue and egg-based media, and in the relatively simple Dubos or Sauton's media showed 8 to 120-fold higher activity in the simpler media.