Recently, a western white pine protein, Pin m III, was shown to be associated with overwintering and frost hardiness of western white pine foliage. To examine whether Pin m III is directly involved in frost hardiness by functioning as an antifreeze protein, work is underway to clone the gene encoding this protein and to assess the function of this gene in freezing tolerance by incorporating the gene in a test plant, such as tobacco. Here, we examined in more detail, by SDS-PAGE and also by two dimensional gel electrophoresis, the seasonal variation of additional proteins in western pine foliage. SDS-PAGE analysis of three seedlots showed that different proteins reached a maximum level in different months, although most proteins (5 to 11) reached a maximum level in winter months (December, January and February). The 2-D gel analysis of foliage sampled on three harvest dates (October, January and April) of one seedlot revealed a seasonal variation of a large number proteins (76 to 184). Of the seasonally varied proteins, the amino terminal sequence of several proteins including Pin m III was determined. One of the sequences was identified by homology to that of the small subunit of ribulose biphosphate carboxylase, whose level increased substantially from fall to spring. The amino terminal sequence of Pin m III had 89% homology to a sugar pine protein, Pin l I. The anti-photosystem II antibody was used to monitor the annual variation of the extrinsic 23-kDa photosystem II protein. The level of the extrinsic 23-kDa photosystem II protein decreased slowly as fall progressed and reached its lowest level in December and then increased in early spring indicating that this variation is due to photosynthetic activity of the foliage during the season.