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Summary: A cold-tolerant line (NY-1) and a cold-sensitive cultivar (Jinyan 4) of cucumber (Cucumis sativus) were treated with temperatures of 28°C/22°C or 28°C/12°C (day/night) in a 10-h photoperiod. Carbohydrates and related enzymes were assayed from 0 to 4h after the start of the dark period. Compared to the normal night temperature (22°C, control), sucrose, stachyose and galactinol increased in mature leaves under cold-night treatment (12°C) while sucrose, glucose and fructose in fruits remained unchanged. In peduncles, where stachyose is catabolized to sucrose after long-distance transport, cold nights simultaneously induced a significant increase of stachyose (substrate) and a decrease of sucrose (product), indicating that the metabolic step from stachyose to sucrose in peduncles is crucial to translocation inhibition in cold nights. This decrease was more pronounced in the cold-sensitive cultivar. Similar growth rates of fruits on one-fruit and two-fruit plants under cold-night treatment further confirmed that it is sink activity rather than source supply that is limiting the source–sink translocation. No significant genotypic differences in enzyme activities involved in the stachyose–sucrose conversion, including alkaline α-galactosidase, acid α-galactosidase, galactokinase, uridine diphosphate (UDP)-galactose pyrophosphorylase, UDP–glucose-4′-epimerase and sucrose synthase, were observed when assayed in an adenosine triphosphate (ATP)-rich in vitro environment. However, the ATP concentration was much higher in peduncles of the cold-tolerant line, indicating that a limiting ATP supply may be partially responsible for the stronger inhibition of the stachyose–sucrose pathway observed in the cold-sensitive cultivar (Jinyan 4). [Copyright &y& Elsevier]