Arabidopsis has a complex and ancient actin gene family encoding six divergent subclasses of proteins. One subclass is represented by ACT2 and ACT8, which encode nearly identical proteins. These two genes differ significantly in flanking and intron sequences and in silent nucleotide positions within codons. Gene-specific RNA gel blot hybridization and reverse transcriptase-mediated polymerase chain reaction (RT-PCR) assays showed that ACT2 and/or ACT8mRNAs were coordinately and strongly expressed in leaves, roots, stems, flowers, pollen, and siliques. Together they account for greater than 80% of the actin mRNA in most Arabidopsis organs. The 5' flanking regions, including the promoter, the mRNA leader exon, an intron in the mRNA leader, and the first 19 codons, were coupled to a beta-glucuronidase (GUS) reporter gene and transformed into Arabidopsis. The ACT2/GUS construct was expressed strongly in nearly all the vegetative tissues in seedlings, juvenile plants, and mature plants. These activities persisted in older tissues. Little or no expression was observed in seed coats, hypocotyls, gynoecia, or pollen sacs. In contrast, the expression of the ACT8/GUS construct was weaker. It was observed only in a subset of the organs and tissues expressing ACT2/GUS and was not significantly expressed in the flower. ACT2, ACT8, and ACT8/GUS mRNAs were present at moderate to high levels in pollen, and yet neither ACT2/GUS nor ACT8/GUS enzyme expression could be detected in pollen. This suggested a mechanism of translational control affecting ACT2 and ACT8 expression in some tissues. The conservation of protein sequence and overlapping patterns of expression, in spite of significant DNA sequence divergence, suggests that the function and regulation of these two genes have been conserved during the evolution of the Brassicaceae.