In Drosophila, dosage compensation-the equalization of most X-linked gene products in males and females-is achieved by a twofold enhancement of the level of transcription of the X chromosome in males relative to each X chromosome in females. A complex consisting of at least five gene products preferentially binds the X chromosome at numerous sites in males and results in a significant increase in the presence of a specific histone isoform, histone 4 acetylated at lysine 16. Recently, RNA transcripts (roX1 and roX2) encoded by two different genes have also been found associated with the X chromosome in males. We have partially purified a complex containing MSL1, -2, and -3, MOF, MLE, and roX2 RNA and demonstrated that it exclusively acetylates H4 at lysine 16 on nucleosomal substrates. These results demonstrate that the MSL complex is responsible for the specific chromatin modification characteristic of the X chromosome in Drosophila males.