To define the transcriptional mechanisms contributing to stage- and tissue-specific expression of muscle genes, we performed transgenic analysis of Drosophila paramyosin gene regulation. This gene has two promoters, one for paramyosin and one for miniparamyosin, which are active in partially overlapping domains. Regions between -0.9 and -1.7 kilobases upstream of each initiation site contribute to the temporal and spatial expression patterns. By comparing the Drosophila melanogaster and Drosophila virilis promoters, conserved binding sites were found for known myogenic factors, including one MEF2 site and three E boxes. In contrast with previous data, our experiments with the paramyosin promoter indicate that the MEF2 site is essential but not sufficient for proper paramyosin gene transcription. Mutations in the three E boxes, on the other hand, do not produce any effect in embryonic/larval muscles. Thus MEF2 site- and E box-binding proteins can play different roles in the regulation of different muscle-specific genes. For the miniparamyosin promoters, several conserved sequences were shown to correspond to functionally important regions. Our data further show that the two promoters work independently. Even when both promoters are active in the same muscle fiber, the transcription driven by one of the promoters is not affected by transcription driven by the other.