In Drosophila, the single male X chromosome is transcribed at twice the rate of a single female X chromosome. This hypertranscription requires the functions of at least four autosomal male-specific lethal genes (msls) and is under the control of the Sex-lethal (Sxl) gene. One of the msls, the maleless (mle) gene, encodes a protein that is associated with the male X chromosome. To investigate how dosage compensation is regulated, we have determined whether Sxl and the other msls are required for mle X chromosome binding. We have found that in females, Sxl functions to prevent mle from binding to the two X chromosomes. Additionally, we have found that mle X chromosome binding requires wild-type msl1, msl2, and msl3 functions. These data support a model whereby the activity of the mle protein is regulated through its association with one or more of the other msl proteins.