Heterosis is broadly defined as the increased vigour of hybrids in comparison to their parents. In the model plant Arabidopsis thaliana, a significant heterosis effect on leaf-freezing tolerance was observed in the F(1) generation of a cross between the accessions Columbia-0 (Col) and C24. Parental Col plants were significantly more freezing-tolerant than C24 plants in both the acclimated and non-acclimated (NA) states. Mid-parent heterosis was observed in the F(1) plants, both in the basic tolerance of non-adapted plants and in freezing tolerance after cold acclimation. Best-parent heterosis, on the other hand, was only found after cold acclimation. The heterosis effect was reduced in the F(2) populations such that only mid-parent heterosis was evident. The leaf content of soluble sugars (fructose (Fru), glucose (Glc), sucrose (Suc) and raffinose (Raf)) increased dramatically in the F(1) plants after cold acclimation as compared to the parental lines. The content of proline (Pro), however, was only moderately increased in the F(1) plants under the same conditions. Correlation analyses showed that only Raf content was consistently related to leaf-freezing tolerance in both the acclimated and NA states. A quantification of mRNA levels in leaves of parental and F(1) lines using quantitative real-time RT-PCR showed no clear indication for an involvement of the investigated genes (CBF (C-repeat binding factor)1, CBF2, (cold-regulated protein (COR) 6.6, COR15a, COR15b, COR47 and COR78) in the heterosis effect.