Oligonucleotides corresponding to highly conserved regions of mammalian protein phosphatase catalytic subunits were used in the polymerase chain reaction (PCR) to generate an amplification product from genomic DNA of Trypanosoma brucei rhodesiense. The PCR product was used to screen a T. b. rhodesiense cDNA library for cDNA clones encoding putative protein phosphatase catalytic subunits. Two cDNA clones, (TPP1A and TPP1B) representing two distinct type 1 catalytic subunit isotypes, encode 39-kDa proteins of 346 amino acids that show 66% and 40% identity, respectively, to mammalian protein phosphatase 1 and 2A catalytic subunits. Both cDNAs are derived from 2.3-kb mRNAs, and Northern blot analysis has provided indirect evidence that these mRNAs are part of the same transcription unit as mRNAs for RNA polymerase II largest subunit. Another cDNA, TPP2, represents the type 2A class of phosphatases and codes for a 34.5-kDa protein of 303 amino acids. The deduced amino acid sequence has 39% and 55% identity, respectively, to the catalytic subunits of mammalian protein phosphatase 1 and 2A. Southern and Northern blot analyses are consistent with TPP2 being encoded by a single copy gene from which is derived a mRNA of 2.5 kb. This finding constitutes the first example in eukaryotes in which a single gene encodes the type 2A class of protein phosphatases. Sera from mice immunized with TPP1A fusion protein reacted with the catalytic subunits of mammalian types 1, 2A and 2B protein phosphatases. However, antisera to TPP2 fusion protein was specific for the type 2A catalytic subunit and recognized a polypeptide of 35 kDa in a Western blot of crude trypanosomal lysate.