Integration of foreign DNA into eukaryotic genomes results frequently in a total or partial loss of gene function, caused by the interruption of indispensable structures of the gene itself. Using T-DNA insertions in Arabidopsis we screened for mutants with deregulated chlorophyll precursor accumulation in etiolated seedlings. A mutant designated rfd1 (red fluorescent in darkness) with increased protochlorophyllide accumulation showed a fluorescent phenotype that was associated with a lack of transcript initiation from the AtRibA1 promoter situated downstream of the integrated T-DNA. Complementation experiments confirmed rfd1 to be a knockout phenotype. Comparison with two SALK insertion lines bearing T-DNA integrations in the 5′UTR of AtRibA1 demonstrated that the insertion event in rfd1 itself does not explain the complete lack of transcript initiation. A 35S tetrameric enhancer sequence present on the rfd1 T-DNA causes the overaccumulation of a large polycistronic transcript originating inside the T-DNA. This 5.5-kb RNA runs over the downstream situated AtRibA1 promoter, which was shown by 5′RACE analyses to be consequently silenced. Hence, a transcription process that starts upstream and overlaps AtRibA1 blocks the initiation at the AtRibA1 promoter in rfd1. This regulatory mechanism has recently been introduced in yeast as transcriptional interference and is described here for the first time in a plant system.