Derivatives of Myxococcus xanthus FB(t) resistant to chloramphenicol (25 mug/ml) arose spontaneously with a frequency of approximately 10(-7). One of these organisms (FB(t)Cam(1) (r)) was characterized. FB(t)Cam(1) (r) showed a unique type of phenotypic instability. After transfer from medium containing chloramphenicol to medium lacking the drug, resistance was lost after approximately one generation. The loss resulted in a sharp drop in the total number of chloramphenicol-resistant organisms and was not due to segregation of chloramphenicol-susceptible organisms during growth. Cell-free extracts of strain FB(t)Cam(1) (r) converted chloramphenicol to acetyl chloramphenicols in a fashion implicating activity of chloramphenicol acetyltransferase. This activity was lost simultaneously with the loss of chloramphenicol resistance after removal of the drug from cultures. Organisms with a similar phenotype to FB(t)Cam(1) (r) could be produced at high frequencies when strain FB(t) was exposed to low concentrations of chloramphenicol (2 to 5 mug/ml), to 3-acetylchloramphenicol (25 mug/ml), or to 1,3-diacetylchloramphenicol (25 mug/ml). Since strain FB(t) is capable of deacetylating acetyl chloramphenicols, these effects are probably all due to low concentrations of chloramphenicol. In the presence of chloramphenicol, FB(t)Cam(1) (r) produced fruiting bodies and myxospores on fruiting agar; however, glycerol-induced myxospore formation was inhibited. In the absence of the antibiotic, chloramphenicol resistance was maintained by glycerol-induced myxospores.