gamma-Butyrobetaine hydroxylase (4-trimethylaminobutyrate, 2-oxoglutarate:oxygen oxidoreductase (3-hydroxylating), EC 1.14.11.1) has been isolated from Pseudomonas sp AK 1 by ion-exchange, adsorption, and molecular-sieving chromatography. The preparation was homogeneous as judged from electrophoresis in agarose and polyacrylamide gels, isoelectric focusing, and equilibrium sedimentation. The molecular mass was 95 kdaltons as determined by sedimentation equilibrium centrifugation. From electrophoresis in polyacrylamide gel the molecular mass was estimated to 92 kdaltons, from gel filtration through columns of Sephadex G-200 to 86 kdaltons, and from gel filtration through thin layers of Sephadex G-150 and G-200 to 82 kdaltons. Calculation of molecular mass from Stokes radius, sedimentation coefficient, and partial specific volume gave a value of 96 kdaltons, and from the sedimentation coefficient, 93 kdaltons. Gel filtration through Sephadex G-200 in 6 M guanidinium chloride and electrophoresis in polyacrylamide gel containing 3.5 mM sodium dodecyl sulfate resulted in single bands with mobilities corresponding to molecular masses of 39 and 37 kdaltons, respectively, indicating that the enzyme is composed of two polypeptides chains with similar size. NH2-terminal amino acid sequencing in three cycles resulted in two amino acids in each cycle (Ala + Asn, Ala + Ile, Ala + Ile). The Stokes radius was 3.8 nm, corresponding to a diffusion coefficient of 5.7 X 10(-7) cm2/s. A sedimentation coefficient of 5.8 X 10(-13) s and a frictional ratio of 1.26 was found. The partial specific volume was 0.729 mL/g at 20 degrees C as calculated from amino acid analysis. The isoelectric point was 5.1, as determined by isoelectric focusing analysis. The light absorption in the ultraviolet and visible regions was that of a protein without light-absorbing prosthetic groups. The absorption coefficient at 280 nm of a 1.0% solution at pH 6.5 was 12.6. Amino acid analysis by ion-exchange chromatography showed a half-cystine content of 19 mol per 95 kg of protein (23 residues/1000). Thirteen sulfhydryl groups were found by colorimetric analysis before as well as after reduction with NaBH4, indicating absence of disulfide bonds. Less than 0.1 mol of iron was found per mol of enzyme.