The enzyme-catalyzed hydration of the citrus by-product, limonene, to the important flavor and aroma chemical, α-terpineol, was investigated. Particulate-associated α-terpineol dehydratase was recovered from Pseudomonas gladioli, solubilized, and partially purified using detergent extraction and gel filtration. Activity of α-terpineol dehydratase was low in non-aqueous solvents. α-Terpineol dehydratase was characterized in buffers containing 0.1% (w/v) Triton X-100. In 10 mM MES, 10 mM BIS-TRIS PROPANE buffer, the pH optimum was 5.5 and the stability optimum was pH 8.0. The temperature optimum at pH 7.0 was 25° in 10 mM HEPES buffer. Using temperature-activity data for 10-25°, Ea and Q10 of α-terpineol dehydratase were determined to be 21.6 ± 2.9 kJ. mol-1 and 1.37 ± 0.07, respectively. Activity was inhibited by Triton X-100. The effects were an increase in apparent Km and decrease in apparent Vmax. Average apparent Km of α-terpineol dehydratase was 2.18 ± 0.19 mM in 10 mM HEPES buffer, pH 7.0 containing 0.1% (w/v) Triton X-100. α-Terpineol dehydratase stereospecifically catalyzed the hydration of (4R)-(+)-limonene to (4R)-(+)-α-terpineol or (4S)-(-)-limonene to (4S)-(-)-α-terpineol. The enzyme was also stereoselective, since the rate of hydration of (4R)-(+)-limonene was approximately ten times faster than the rate of hydration of (4S)-(-)-limonene.