We have isolated actin genes from genomic libraries of two highly diverged plants, maize and soybean. The complete nucleotide sequences of a maize actin gene, MAc1, and a soybean actin gene, SAc1, were determined. The nucleotide sequences of these two actin genes and of a previously sequenced soybean actin gene were compared with the actin gene sequences from a wide spectrum of evolutionarily diverged eukaryotes. Some striking features pertinent to the evolution and function of the plant actin gene families have emerged. The deduced amino acid sequence of the plant actins resembles both cytoplasmic- and muscle-specific actins. DNA sequence analysis as well as genomic blotting experiments using cloned actin sequences as probes show that large sequence heterogeneity exists among members of the plant actin multigene families and between genes from two highly diverged plant species. The sequences of the first nine amino acids at the amino terminal end of the plant actins are far more conserved between distant plant actins than the corresponding sequences in distantly related animal actin genes, suggesting a unique and conserved function for the NH2 terminal sequence in higher plants. The soybean and maize actin genes examined each contain three introns in precisely the same positions, quite contrary to the divergent placement of introns observed in animal, protozoan, and fungal actins. The position of the first intron in soybean and maize actin genes corresponds precisely to the position of an intron found in a nematode actin gene. The position of the second intron coincides with one found in rat and chicken skeletal actin genes. These data suggest that the numerous introns found in all actins are of ancient origin. The degree of silent substitution and replacement substitution was compared among plant actin genes and to those of animal, protozoan, and yeast actin genes. It is clear that the silent substitution sites are saturated among all the genes compared, whereas the replacement sites have diverged in only 5-17% of their possible positions. By these criteria the most distant animal actins are only 6% diverged. The three plant actin genes examined are 8-10% diverged in replacement sites from each other and approximately 14% diverged in replacement sites from any of the animal actins examined. The data in this manuscript suggest that the families of soybean and maize actin genes may have diverged from a single common ancestral actin gene long before the divergence of monocots and dicots.