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Strawberry, belonging to the Rosaceae family, translocates sucrose as the main carbohydrate to fruit, although many Rosaceae fruit trees use sorbitol as translocated sugar; however, we have found that strawberry has genes encoding sorbitol-metabolizing enzymes. In this study, the property of NAD^+-dependent sorbitol dehydrogenase (NAD-SDH) and the effect of sugars and phytohormones on NAD-SDH activity and its mRNA accumulation were investigated to elucidate sorbitol metabolism and its regulation. The K_m values of NAD-SDH for substrates of fructose and sorbitol were 78.7 and 7.3 mM, respectively, similar to those of maize, which synthesizes sorbitol by the reduction of fructose by NAD-SDH. This result suggested that NAD-SDH in strawberry fruit can catalyze the reduction of fructose, but the activity is not enough to accumulate sorbitol in fruit. Therefore, we investigated the effect of sugars and phytohormones on NAD-SDH activity and the transcript level by adding various sugars and phytohormones to sliced fruit discs. Tissues incubated in 100 mM fructose or sorbitol stimulated NAD-SDH activity by about 2.5-fold compared with the control. Sucrose also stimulated NAD-SDH activity, but the increase was not as high as sorbitol or fructose. Of the phytohormones treated, 100 μM indole-3-acetic acid (IAA) had a marked stimulatory effect on NAD-SDH activity, but the other phytohormones (abscisic acid, gibberellic acid and 6-benzyladenine) had no stimulative effect. The mRNA transcript level in all sugar and phytohormone treatments showed no marked increase compared with the control. Thus, the kinetics and induction profiles by phytohormones of NAD-SDH showed that sorbitol metabolism in strawberry fruit was different from that in apple and Japanese pear fruits.