The non-coding roX1 and roX2 RNAs are components of the MSL dosage compensation complex in Drosophila. We found that multiple species of roX2 RNA are produced by alternative splicing, with one major and at least 20 different minor forms associated with MSL proteins. The alternative forms are generated by variable usage of multiple 5' and 3' splice sites between two common exons. This alternative splicing is evolutionarily conserved in several distant Drosophila species in spite of differences in primary sequences. Transgenic constructs expressing individual major or minor D. melanogaster roX2 species display low steady-state levels of roX2 RNA, weak accumulation of MSL complex on the X chromosome, and low rescue of male-specific roX(-) lethality. Increased expression of individual roX2 forms using the constitutive Hsp83 promoter results in increased transgenic rescue of roX(-) mutant male flies. However, although males survive they are delayed in their development. In addition, MSL complexes still show low affinity for the X chromosome and abnormal accumulation at the transgenic site of synthesis of the individual roX2 alternative splice form. Taken together, these results suggest an important role for roX2 RNA splicing in optimal MSL complex assembly or function.