We have identified four mutations affecting the actin III isoform in the indirect flight muscles (IFM) of Drosophila. One mutation does not produce any protein product, and three direct the synthesis of electrophoretic variants of actin. Complementation tests and recombination mapping indicate that all mutations are alleles of an actin gene at chromosomal band 88F (act88F gene). The effect of these mutations is restricted to the IFM. We conclude that the act88F gene is expressed only in the IFM to encode actin III, which is its major isoform. In two of the actin mutants, heat shock proteins are constitutively expressed in the IFM. Genetic evidence strongly suggest that this anomaly is primarily caused by the mutations in the act88F structural gene.