Sixteen bacterial strains isolated from soil were able to grow on either 2-monochloropropionic acid or monochloroacetic acid as the sole carbon and energy source. The isolates were divided into five groups on the basis of differences in their dehalogenase activities towards four substrates — monochloroacetic acid, dichloroacetic acid, 2-monochloropropionic acid and 2,2-dichloropropionic acid. Disc gel electrophoresis of crude extracts identified four distinct dehalogenases with different electrophoretic mobilities: three isolates contained one, three or four dehalogenases, respectively, and the remaining 13 isolates contained different combinations of two dehalogenases. In some cases, dehalogenases with the same mobilities from different isolates appeared to be identical enzymes. In others, enzymes from different isolates with the same electrophoretic mobility had different substrate activity profiles. Pseudomonas putida PP3 was shown to contain one enzyme which was comparable with one of the dehalogenases detected in several of the newly isolated soil bacteria. The second enzyme was not found in soil bacteria and represented a fifth dehalogenase. The significance of these results in terms of the evolution of dehalogenase activity is discussed.