The male-specific lethal (MSL) ribonucleoprotein complex is necessary for equalization of X:A expression levels in Drosophila males, which have a single X chromosome. It binds selectively to the male X chromosome and directs acetylation of histone H4 at lysine 16 (H4Ac16), a modification linked to elevated transcription. roX1 and roX2 noncoding RNAs are essential but redundant components of this complex. Simultaneous removal of both roX RNAs reduces X localization of the MSL proteins and permits their ectopic binding to autosomal sites and the chromocenter. However, the MSL proteins still colocalize, and low levels of H4Ac16 are detected at ectopic sites of MSL binding and residual sites on the X chromosome of roX1- roX2- males. Microarray analysis was performed to reveal the effect of roX1 and roX2 elimination on X-linked and autosomal gene expression. Expression of the X chromosome is decreased by 26% in roX1- roX2- male larvae. Enhanced expression could not be detected at autosomal sites of MSL binding in roX1- roX2- males. These results implicate failure to compensate X-linked genes, rather than inappropriate upregulation of autosomal genes at ectopic sites of MSL binding, as the primary cause of male lethality upon loss of roX RNAs.