Evolutionary and immunological relationships among the different genes specifying synthesis of rainbow trout (Salmo gairdneri) LDH subunits were demonstrated. A procedure was devised for the purification and concentration of specific rainbow trout isozymes which were then used for antisera production. The B41and B42autotetramer isozymes and the Group “a” isozyme system were shown by double agar diffusion to produce distinctly different antibodies. Antisera against specific isozymes showed degrees of cross-reaction depending on the relatedness of the isozymes. The LDH-C gene locus is more closely related to the LDH-B2gene than to the LDH-B1gene. Electrophoresis was demonstrated to be an efficient additional method to spectrophotometric assays to show inactivation of LDH by antibodies. LDH isozymes underwent rapid conformational change when purified and stored.