As a first step toward developing a system of genetic exchange between Vibrio parahaemolyticus strains, spontaneously arising auxotrophic and Kanagawa phenomenon-negative (KP-) mutants were isolated and characterized. Auxotrophic mutants were selected by nalidixic acid enrichment of parental cultures. Some Cys- and Arg- mutants of a KP+ strain were found to be KP-. Reversion to prototrophy by these strains was not accompanied by a return to the parental KP+ phenotype. Additionally, two prototrophic KP- mutants were isolated. No detectable levels of vibriolysin were found in supernatant extracts of KP- mutants by slide gel immunodiffusion analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, or assay for lethal activity in mice. All Cys-, Arg-, and Pur- mutants tested reverted to a different auxotrophy (phenotypic interconversion) as well as to prototrophy. The possible role of insertion sequence-like elements in vibriolysin production and phenotypic interconversion is discussed.