Actin genes in Arabidopsis thaliana exist as an ancient gene family. A. thaliana actins comprise two ancient classes, reproductive and vegetative. ACT1 and ACT3, the result of a duplication event 30-60 MYA, comprise one of the five ancient subclasses and both are expressed at high levels in mature pollen, embryo sac, and organ primordia. ACT1 and ACT3 share little homology with exception of a 55 base pair sequence within the 5' flanking region. A fluorescence assay measuring reporter gene expression was designed to quantify changes in expression in various promoter/GUS fusion constructs. Truncations of the 5' upstream flanking region, within an adjacent intron, and site-directed mutagenesis constructs demonstrate that all these regions are needed to promote high levels of mature pollen expression. The first intron in ACT1 is required for high levels of pollen expression and organ primordia expression. This intron functions in a gene-specific manner as substitution of the first intron of ACT1 with the first intron of a vegetatively expressed actin efficiently down-regulates pollen expression. Multiple sequence elements within the ACT1 5' flanking region direct high levels of mature pollen specific and organ primordia expression. |Ancient classes of actin could have been preserved because their expression was under strong selective constraint. Cis-elements and transcription factors may function in distant plant species. 5' flanking regions of the ACT1 gene translationaly fused to a reporter gene were transformed into distantly related Nicotiana tabacum and Oryza sativa. ACT1 was expressed almost identically in N. tabacum and O. sativa as in A. thaliana. The heterologous transformation results clearly show that the expression patterns of the reproductive classes of actin genes are highly conserved in heterologous species in reproductive organs, but not vegetative organs. Both traditional methods of promoter dissection and examination of gene expression within a heterologous system are useful in analyzing duplicated regulatory regions throughout the evolution of the actin gene family.