This report describes a method for the easy generation of inverted repeat constructs for the silencing of genes of unknown sequence which is applicable to high-throughput studies. This improved procedure for high-efficiency gene silencing is specific for a target gene, but does not require inverted repeat DNA of the target gene in the construct. The method employs an inverted repeat of the 3'-untranslated region (3'-UTR) of a heterologous gene, and has been demonstrated using the 3'-UTR region of the nopaline synthase (nos ) gene from Agrobacterium tumefaciens , which is often used as the 3'-UTR for transgene constructs. In a population of independent tomato primary transformants harboring a stably integrated polygalacturonase (PG ) transgene driven by a constitutive promoter and linked to an inverted repeat of the nos 3'-UTR, 51 of 56 primary transformants (91% of the population) showed highly effective post-transcriptional silencing of the PG gene, with PG mRNA abundance in ripe fruit reduced by 98% or more. The method was also effective in Arabidopsis , where two different, relatively uncharacterized plant transcription factors were also targeted effectively. This method has the advantage of ease and rapidity in preparation of the constructs, since a gene of interest can be inserted into a binary vector already containing the promoter and the inverted nos domain in a single-cloning step, and does not require any knowledge of the DNA sequence. The approach is suitable for high-throughput gene silencing studies, where it is necessary to investigate the function of hundreds to thousands of uncharacterized genes.