Actin is a highly conserved protein found in all eukaryotic organisms. Most organisms have multiple cytoplasmic actin genes that encode isoforms with slightly different amino acid sequences. These different isoforms are coexpressed in many cell types. Why organisms have multiple very similar cytoplasmic actin genes is unclear. We have addressed this question with the cytoplasmic actins in Drosophila, Act5C, and Act42A. These isoforms differ by only two amino acids and both genes are expressed in all cells at all times during development. We identified P element insertions in the Act5C gene that resulted in a lethal phenotype. The lethal phenotype is rescued by a transgene with a genomic fragment that includes Act5C regulatory and amino acid coding sequences. A hybrid transgene containing the protein coding sequence for the Act42A isoform, under the control of the regulatory regions of the Act5C gene, also rescues the lethality of the Act5C mutants. Furthermore, flies that carry only one copy each of Act5C and Act42A are viable. These results suggest the amino acid differences between these two cytoplasmic actin isoforms are not important for function and the need for increased gene dosage to provide more actin is not likely to explain the existence of multiple genes. Instead, our results suggest that regulated expression of Act5C is essential to the fly.