Immunostaining of chromosomes shows that the male-specific lethal (MSL) proteins are associated with all female chromosomes at a low level but are sequestered to the X chromosome in males. Histone-4 Lys-16 acetylation follows a similar pattern in normal males and females, being higher on the X and lower on the autosomes in males than in females. However, the staining pattern of acetylation and the mof gene product, a putative histone acetylase, in msl mutant males returns to a uniform genome-wide distribution as found in females. Gene expression on the autosomes correlates with the level of histone-4 acetylation. With minor exceptions, the expression levels of X-linked genes are maintained with either an increase or decrease of acetylation, suggesting that the MSL complex renders gene activity unresponsive to H4Lys16 acetylation. Evidence was also found for the presence of nucleation sites for association of the MSL proteins with the X chromosome rather than individual gene binding sequences. We suggest that sequestration of the MSL proteins occurs in males to nullify on the autosomes and maintain on the X, an inverse effect produced by negatively acting dosage-dependent regulatory genes as a consequence of the evolution of the X/Y sex chromosomal system.