The Saccharomyces cerevisiae type 2A protein phosphatase (PP2A) Pph22p differs from the catalytic subunits of PP2A (PP2Ac) present in mammals, plants and Schizosaccharomyces pombe by a unique N-terminal extension of approximately 70 amino acids. We have overexpressed S. cerevisiae Pph22p and its N-terminal deletion mutant Delta N-Pph22p in the GS115 strain of Pichia pastoris and purified these enzymes to apparent homogeneity. Similar to other heterologous systems used to overexpress PP2Ac, a low yield of an active enzyme was obtained. The recombinant enzymes designed with an 8 x His-tag at their N-terminus were purified by ion-exchange chromatography on DEAE-Sephacel and affinity chromatography on Ni2+-nitrilotriacetic acid agarose. Comparison of biochemical properties of purified Pph22p and Delta N-Pph22p with purified human 8 x His PP2Ac identified similarities and differences between these two enzymes. Both enzymes displayed similar specific activities with 32P-labelled phosphorylase a as substrate. Furthermore, selected inhibitors and metal ions affected their activities to the same extend. In contrast to the mammalian catalytic subunit PP2Ac, but similar to the dimeric form of mammalian PP2A, Pph22p, but not Delta N-Pph22p, interacted strongly with protamine. Also with regard to the effects of protamine and polylysine on phosphatase activity Pph22p, but not Delta N-Pph22p, behaved similarly to the PP2Ac-PR65 dimer, indicating a regulatory role for the N-terminal extension of Pph22p. The N-terminal extension appears also responsible for interactions with phospholipids. Additionally Pph22p has different redox properties than PP2Ac; in contrast to human PP2Ac it cannot be reactivated by reducing agents. These properties make the S. cerevisiae Pph22p phosphatase a unique enzyme among all type 2A protein phosphatases studied so far.