SummaryThe first intron of the petunia actin‐depolymerizing factor 1 (PhADF1) gene was previously shown to induce strong and constitutive expression of that gene in vegetative tissues of transgenic Arabidopsis. To examine intron‐mediated enhancement of PhADF1 gene expression in detail, the effects of splicing, deletion and promoter alteration on gene expression were analyzed in this study. Deletion of the 5′ upstream region of the intron significantly reduced the level of enhancement, under the control of both the PhADF1 and the PhADF2 promoters. The ratio of pre‐mRNA and mRNA does not correlate with the level of enhancement. To determine whether there is a promoter–intron interaction, the role of the intron was examined under the control of a heterogeneous promoter. The intron of PhADF1 induced GUS expression in vegetative tissues under the control of the reproductive tissue‐specific Arabidopsis profilin 5 (PRF5) promoter. In transient assays, the presence of the intron increased GUS expression under control of the 35S minimal promoter. Our results suggest that the first intron of the PhADF1 gene alters tissue‐specific expression by a post‐transcriptional mechanism. In addition, we have also shown that intron‐mediated enhancement is a conserved mechanism, which regulates the expression of the petunia and Arabidopsis ADF genes that are expressed in vegetative tissues.