Experiments were performed to demonstrate the production in E. coli of spontaneously occurring mutants resistant to proflavine. The fluctuation test of Luria and Delbruck suggested that such mutants arise. This was confirmed by a modification of the replica plating technique of Lederberg and Lederberg. By this method, 2 separate first-step mutants were isolated; from one of these a 2nd-step mutant was isolated, and from this a 3d-step mutant. The resistance of the 3d-step mutant was about 100 times greater than that of the original sensitive strain. By the same technique, 2 first-step mutants to chloramphenicol resistance were isolated. None of the 6 mutants had come into contact with either drug until the time of testing. Cross-resistance between proflavine and chloramphenicol was shown by 5 of the 6 mutants; the 6th showed an increase in proflavine resistance though not in resistance to chloramphenicol.