A protocol is described for the first isolation of salt-sensitive mutants from a moderately halophilic eubacterium, namely Vibrio costicola, which normally can grow in up to 3.4 M NaCl. Four groups of mutants, which are all unable to grow at high (i.e., 2.5-3.0 M) concentrations of NaCl, have been characterized on the basis of their salt and temperature sensitivities. The acquisition of these mutants will be helpful in clarifying the role of membrane lipid changes in haloadaptation.