Seedlings of Scots pine (Pinus sylvestris L.), with only primary needles, were frost hardened under a photoperiod of 8 h and a temperature of 4 °C for 6–9 weeks. The effects of frost hardening were studied on (i) the photosynthetic efficiency as defined by the quantum yield of CO2 uptake under light-limiting conditions, (ii) the energy partitioning between the two photosystems as analyzed by 77 K fluorescence spectroscopy, and (iii) the distribution of solubilized chlorophyll–protein complexes after electrophoretic separation. It was found that frost hardening had no significant effects either on the photosynthetic quantum yield measured at 23–25 °C or on the energy distribution between the two photosystems. Despite these findings, frost hardening affected the chlorophyll organization so that the proportion of aggregated complexes such as chlorophyll–protein 1a and the dimeric form of light-harvesting chlorophyll–protein decreased. Frost hardening also decreased the amount of chlorophyll–protein a and increased the amount of free, solubilized chlorophyll in the gel scan profile. From these results it is concluded that frost hardening affects the chemical interactions within the antennae organization of the photosynthetic apparatus but that these changes have no significant effects on the energy distribution between the two photosystems or on the photosynthetic efficiency as defined by the quantum yield for CO2 uptake.