Type II fish antifreeze protein (AFP) is active in both freezing point depression and the inhibition of ice recrystallization. This extensively disulfide-bonded 14 kDa protein was targeted for accumulation in its pro- and mature forms in the cytosol and apoplast of transgenic tobacco plants. Type II AFP gene constructs under control of a duplicate cauliflower mosaic virus 35S promoter, both with and without a native plant transit peptide sequence, were introduced into tobacco by Agrobacterium tumefaciens-mediated transformation. AFP did not accumulate in the cytosol of transgenic plants, but active AFP was present as 2% of the total protein present in the apoplast. Plant-produced AFP was the same size as mature Type II AFP isolated from fish, and was comparable to wild-type AFP in thermal hysteresis activity and its effect on ice crystal morphology. Field trials conducted in late summer on R1 generation transgenic plants showed similar AFP accumulation in plants under field conditions at levels suitable for large-scale production: but no difference in frost resistance was observed between transgenic and wild-type plants during the onset of early fall frosts.