Protein phosphatase type 1 (PP1) is one of the major classes of serine/threonine protein phosphatases, and has been found in all eukaryotic cells examined to date. Metazoans from Drosophila to humans have multiple genes encoding catalytic subunits of PP1 (PP1c), which are involved in a wide range of biological processes. Different PP1c isoforms have pleiotropic and overlapping functions; this has complicated the analysis of their biological roles and the identification of specific in vivo substrates. PP1c isoforms are associated in vivo with regulatory subunits that target them to specific locations and modify their substrate specificity and activity. The PP1c-binding proteins are therefore the key to understanding the role of PP1 in particular biological processes. The existence of isoform specific PP1c-binding subunits may also help to explain the unique roles of different PP1c isoforms. Here we report the identification of 24 genes encoding Drosophila PP1c-binding proteins in the yeast two-hybrid system. Sequence analysis identified a minimal interacting fragment and putative PP1c-binding motif for each protein, delimiting the region involved in binding to PP1c. Further two-hybrid analysis showed that virtually all of the interactors were capable of binding all Drosophila PP1c isoforms. One of the novel interactors, CG1553, was examined further and shown to interact with multiple isoforms by co-immunoprecipitation from Drosophila extracts and functional interaction with PP1c isoforms in vivo. Bioinformatic analyses implicate the putative PP1c-associated subunits in a diverse array of intracellular processes. Our identification of a large number of PP1c-binding proteins with the potential for directing PP1c's specific functions in Drosophila represents a significant step towards a full understanding of the range of PP1 complexes and function in animals.