Chloramphenicol-nonproducing and plasmid-less mutants obtained previously by treatment with acriflavine still produced a small amount of chloramphenicol in a medium. To study the role of plasmid in chloramphenicol production, 70 chloramphenicol-nonproducing mutants were isolated by acriflavine treatment, high-temperature incubation, UV-irradiation or nitrosoguanidine treatment, starting from a producer (SVM2). Most of them did not produce any amount of chloramphenicol. One mutant, SVM2-2A7 was found to produce 1-deoxychloramphenicol instead of chloramphenicol. The mutations (cpp) affecting chloramphenicol production were analyzed by crosses with a producing strain carrying the complementing auxotrophic markers. Except for the plasmid-less strains, all Cpp mutations including the 1-deoxychloramphenicol-producing mutation were mapped between met and ilv on the chromosome. Additional crosses indicated that these chromosomal cpp mutants still carried the plasmids which had a role in increasing chloramphenicol production. Therefore, it can be concluded that the structural genes for all or most steps of chloramphenicol biosynthesis including the 3-hydroxylation of p-aminophenylalanine are located between met and ilv on the chromosome of S. venezuelae and that the plasmid plays an important role in increasing the chloramphenicol production. The activity of arylamine synthetase involved in the initial step of the chloramphenicol biosynthesis was unrelated to the presence or absence of plasmid. Moreover, the presence of plasmids was not required for host resistance to chloramphenicol.