The development of the indirect flight muscles of Drosophila melanogaster was studied by analysing mutations that cause flightlessness. Twenty-five mutations on the X-chromosome and two on the third chromosome were examined. The X-chromosomal mutations form ten complementation units. The ten loci were assigned preliminary map positions by meiotic recombination and deficiencies and duplications. The two autosomal mutations represent two genes. Gynandromorph analyses suggest that many of these mutations have their primary effect in the presumptive thoracic muscle region of the embryo. The mutations cause a variety of characteristic defects, such as absence of the bulk of the thoracic muscle mass, or absence of only one of the two fibrillar muscle groups. Electronmicroscopic studies of sixteen mutants representing all twelve loci reveal abnormal myofibrillar organization in some of these mutants, e.g. aberrant or missing Z-bands, or absence of the thin filaments. Mutant protein patterns, obtained by SDS-polyacrylamide gel electrophoresis, show the following differences from wild type: ten mutants are characterized by absence of reduction of the 54 K protein, and most mutants exhibit a reduction and modification of the 80 and 90 K proteins. The absence of reduction of the 54 K protein was strongly correlated with aberrant Z-bands.