Abstract The majority of spontaneous his + revertants of a his − threo + strain of Bacillus subtilis arose on the plates. Few, if any, such revertants arose when a his − threo − strain was plated on the same selective medium with an additional 30 μg/ml of threonine. An inhibitory role of this supplement on the formation of his + revertants was excluded. The inhibitory effect on spontaneous his + reversion thus seemed to be due to the threonineless genetic background. Further analysis, however, revealed that it was due to an interaction, not previously reported, between genetic background and medial factors. It was shown that concentrations of threonine which allowed normal growth of the his − threo − strain were not sufficient for the growth of his + reversions, most of which arose late on the plates. Addition of excess threonine allowed his + reversions to develop. It will be necessary to consider the possibility of such an auxotrophic preemption of medial growth factors in mutation experiments with multiple auxotrophic strains.