Phenylalanine ammonia-lyase (PAL) induction in UVB-exposed plants leads to an increased synthesis of UV-absorbing phenols. As phenols, including anthocycanins, are linked to many protective mechanisms in plants, we tested the hypothesis that UVB-induced phenol accumulation, mediated by PAL, may confer freezing tolerance in jack pine (Pinus banksiana Lamb) seedlings. The hypothesis was tested by applying UVB in the presence and absence of the PAL-inhibitor, 2-aminoindan-2-phosphonic acid (AIP). Jack pine seedlings were grown for 3 weeks with and without 10 µM aqueous AIP. Each treatment was then divided into two groups. One group received near-ambient UVB (5.5 kJ m−2 day−1of biologically effective radiation) for up to 30 h. A second, control group of seedlings received no UVB. Anthocyanin concentration declined by > 99% in PAL-inhibited seedlings and other methanol-extractable UV-absorbing phenols declined by > 48%, relative to the controls. A 20-h exposure to UVB increased seedling freezing (−15°C) tolerance in the absence of the PAL-inhibitor, as shown by a 30% reduction in membrane injury, determined by electrolyte leakage measurements. In PAL-inhibited seedlings, by contrast, the same UVB pre-treatment increased freezing injury by 48%. A longer (30 h) UVB exposure was damaging to both AIP-treated and untreated seedlings. Root feeding with 10 µM AIP during a 3-week exposure of older (6-month-old) seedlings similarly reduced phenol accumulation in UVB-exposed seedlings. The decline in phenol production in PAL-inhibited seedlings correlated with increased freezing injury. These results suggest a role for ambient UVB in seedling frost hardiness, mediated by a PAL-induced production of phenolic compounds.