A gram-negative bacterium that grows on n-decanol was isolated from a sandy beach soil sample. This organism, designated K23-1, grew on media containing various n-alkanols of chain length between C-2 and C-16 at 30 degrees C. On the basis of results of biochemical and morphological tests and sequence analysis of gyrB (the structural gene for the DNA gyrase beta-subunit) and 16S rRNA, the strain was identified as Pseudomonas putida. P. putida K23-1 constitutively expressed NAD(+)-dependent long-chain n-alkanol dehydrogenase. This enzyme was purified about 40-folds to the homogeneity by anion-exchange chromatography followed by hydrophobic interaction chromatography with a yield of about 5%. The molecular mass of the enzyme was determined to be 40 kDa by electrophoresis, and 163 kDa by gel-filtration analysis. The N-terminal amino acid sequence of the enzyme was 50% homologous to that of alcohol dehydrogenase I from Zymomonas mobilis. The enzyme was found to contain 1 mol/mol of zinc. Thus, this long-chain n-alkanol dehydrogenase belonged to the superfamily of Zn-dependent alcohol dehydrogenases. The enzyme was capable of oxidizing n-alkanols of chain length C-2 to C-12, isopropanol and benzyl alcohol. The specificity constants (V-max/K-m) of the enzyme were almost the same for n-alkanols of chain length between C-3 and C-10. The enzyme also catalyzed the reverse reaction. The optimum pH for the forward reaction with n-decanol was 9.5, and that for the reverse reaction with n-decanal was 7.0. The optimum temperature for the forward reaction was 50 degrees C.