We have previously distinguished two different types of resistance response, complete and delayed, to the pepper mild mottle tobamovirus (PMMoV) in Nicotiana benthamiana plants transformed with the 54-kDa region of the viral replicase gene. The present study was undertaken to assess a potential relationship between transgene dose and the type of resistance observed in the 54-kDa transgenic plants. By genetic and molecular analysis, we demonstrated that complete resistance against PMMoV infection was strictly correlated with homozygosity of the transgene in the R1 and R2 generation plants. Progeny hemizygous for the T-DNA locus showed delayed resistance, but none of the progeny exhibited the complete resistance response. Thus, the resistance to PMMoV varied in a gene dosage-dependent manner. However, a limited number of individuals of the R2 progeny from homozygous plants displayed complete resistance, while the remaining plants showed delayed resistanc; the ratio between the two resistance responses was roughly similar to the ratio observed in either the R2 progeny from hemizygous plants or the original R1 generation plants. R2 homozygous plants displaying complete resistance contained undetectable levels of 54-kDa mRNA, although the transgene was accurately transcribed in these plants. These results suggest that transgene homozygosity is a necessary but not a sufficient condition to confer complete resistance to PMMoV by a gene silencing mechanism.