Gene silencing by an ACO1 [1-aminocyclopropane-1-carboxylate (ACC) oxidase 1] sense transgene in tomato plants was correlated with the production of small antisense RNAs (asRNAs) of 21-28 nucleotides, which were preferentially generated from the 3' region of the transgene. Adding inverted repeats (IRs) to the 5' untranslated region of the ACO1 transgene led to stronger silencing than was obtained with the transgene lacking the IRs, and in these plants the asRNAs were preferentially produced from the 5' region, including the IRs themselves and sequences immediately downstream. This observation indicates that secondary structure, including inverted repeats, may be a key determinant of small RNA production in gene silencing. Small asRNAs of 28 nt were much more abundant in the line containing the IRs than in the line without IRs, and may contribute to the stronger silencing associated with the IRs. Much lower levels of small RNA species were detected in plants containing an antisense ACO1 transgene than in an ACO1-sense silenced line showing weaker silencing. This suggests that the stronger suppression of the endogenous ACO1 gene by an antisense transgene may be the result of the combined effects of large antisense RNAs produced from the antisense transgene and small asRNAs.