Modification of tree species through genetic transformation is dependant upon the stability of expression of the introduced gene (transgene). While cases of 'gene silencing' have been reported in herbaceous plants relatively little information is available for trees. In order to investigate the occurrence of such events in trees seventy hybrid poplar (INRA clone 717 1-B4, Populus tremula x P. alba) lines have been independently transformed with the GUS reporter gene under the control of either a constitutive promoter (35S) or a tissue-specific promoter (EuCAD cinnamyl alcohol dehydrogenase from Eucalyptus gunnii Hook.). Histochemistry and spectrofluorometry are being used to follow the stability of transgene expression in stem/branch samples under in vitro, greenhouse and field conditions throughout the year in twenty of these lines. Histochemical results indicate that transgene expression is stable with regard to tissue specificity while spectrofluorometric measurements show that the average transgene expression level decreases significantly upon transfer from in vitro to greenhouse conditions. A case of 'gene silencing' has been observed in one 35S-GUS transformant and three different individuals no longer express the transgene following transfer to field conditions. Molecular analysis is underway to investigate the level (transcriptional vs post-transcriptional) at which silencing has occurred. The effects of the stressing agent 5-propionic acid on transgene expression in vitro is being investigated in both 35S-GUS and EuCAD-GUS lines, as is the effect of the demethylating agent 5-azacytidine on one 35S-GUS transformant which does not express the transgene in vitro.