Control of protein phosphatases is now understood to depend on binding to a variety of regulatory or targeting subunits to form holoenzymes with restricted localization and substrate specificity. In addition, the catalytic subunits of both type-1 and type-2 phosphatases bind specific inhibitor proteins. Here, we report discovery of a new inhibitor protein called PHI-1 that is specific for type-1 protein phosphatase (PP1). Recombinant tagged PHI-1 was phosphorylated by protein kinase C at two sites, one a Ser and one a Thr; phosphorylation enhanced inhibitory potency 50-fold. Mutation of Thr57 to Ala gave a protein phosphorylated only on Ser, without change in inhibitory activity, indicating that phosphorylation of Thr57 was required for full activity. Immunoblotting showed that PHI-1 was expressed in most animal tissues and several cell lines, and a second larger protein called PHI-2 was present in different muscles, especially cardiac muscle. Unlike any other known inhibitor, PHI-1 inhibited the myosin- and glycogen-associated holoenzyme versions of PP1 as well as the monomeric catalytic subunit of PP1. Discovery of PHI-1 and PHI-2 opens new possibilities for regulation of PP1 via phosphorylation-dependent signaling pathways.