Extensive methylation of the Ubi1 promoter has been shown to be associated with transcriptional silencing and bialaphos herbicide sensitivity in several R1 progeny derived from a transgenic rice line, JKA 52, containing multiple copies of the introduced genes (Kumpatla et al., 1997; Plant Physiol. 115, 361-373). All of the progeny from silenced R1 lines were silenced, and a proportion (24%) of the R2 seedlings from JKA 52-6, a non-silenced homozygous R1 line, were found to be silenced. Several nonsilenced (bialaphos-resistant) R2 progeny derived from the selfed seed of JKA 52-6 were tested at various stages throughout the growth period and found to be resistant to bialaphos, indicating the absence of silencing. Five of these were investigated further to determine whether silencing could recur in subsequent generations. R3 seedlings were raised from selfed R2 seed and herbicide sensitivity was detected in 5-40% of the plants in three of the five lines. This silencing was accompanied by methylation of the Ubi1 promoter and the absence of steady-state bar mRNA. This recurrent onset of silencing and instability of expression associated with a transgene locus containing several repeated sequences is undesirable. The data strongly suggest that multi-generation molecular analysis should be undertaken for transgenic lines to be used for agricultural purposes and that lines containing methylated inserts should be carefully evaluated before being included in further breeding strategies.