The SIN3 corepressor serves as a scaffold for the assembly of histone deacetylase (HDAC) complexes. SIN3 and its associated HDAC have been shown to have critical roles in both development and the regulation of cell cycle progression. Although multiple SIN3 isoforms have been reported in simple to complex eukaryotic organisms, the mechanisms by which such isoforms regulate specific biological processes are still largely uncharacterized. To gain insight into how SIN3 isoform-specific function contributes to the growth and development of a metazoan organism, we have affinity-purified two SIN3 isoform-specific complexes, SIN3 187 and 220, from Drosophila S2 cells and embryos. We have identified a number of proteins common to the complexes, including the HDAC RPD3, as well as orthologs of several proteins known to have roles in regulating cell proliferation in other organisms. We additionally identified factors, including the histone demethylase little imaginal discs and histone-interacting protein p55, that exhibited a preferential interaction with the largest SIN3 isoform. Our experiments indicate that the isoforms are associated with distinct HDAC activity and are recruited to unique and shared sites along polytene chromosome arms. Furthermore, although expression of SIN3 220 can substitute for genetic loss of other isoforms, expression of SIN3 187 does not support Drosophila viability. Together our findings suggest that SIN3 isoforms serve distinct roles in transcriptional regulation by partnering with different histone-modifying enzymes.