l-Carnitine dehydrogenase (CDH) is as an excellent tool for l-carnitine (l-Car) estimation. To date, four CDHs have been identified, that share 45 % homology of their proteins. Here 42 conserved residues of CDH from Xanthomonas translucens (Xt-CDH) were substituted successively with alanine. The resultant mutants were analyzed for catalytic activity. Active mutants were evaluated for their influence on l-Car affinity. Twenty-three mutants with reduced affinity toward l-Car were subjected to detailed kinetic analysis. Analytical data implied that all mutants had increased K (m) values. The mutants of R193A, E196A, W199A, R200A, F249A, and F253A that produced the greatest l-Car affinity disruption (K (m) > 200-folds of Xt-CDH) clustered near the putative active site. This information can provide a solid basis for the rational design of mutagenic investigation to improve CDHs.