The complete macronuclear DNA polymerase alpha gene, previously sequenced in Oxytricha nova, has been cloned from a genomic macronuclear library and sequenced for the hypotrich O. trifallax. Macronuclear DNA clones of DNA polymerase alpha encoding approximately 1000 amino acids, or approximately two-thirds of the open reading frame, have been obtained by PCR and sequenced for Halteria grandinella, Holosticha species, Paraurostyla viridis, Pleurotricha lanceolata, Stylonychia lemnae Teller, Sty. mytilus, Uroleptus gallina, and Urostyla grandis. Phylogenetic relationships inferred from DNA polymerase alpha amino acid sequences have been used to clarify taxonomic relationships previously determined by morphology of the cell cortex. Hypotrich phylogenies based on DNA polymerase alpha amino acid sequences are incongruent with morphological and other molecular phylogenies. Based upon these data, we assert that, contrary to morphological data, O. nova and O. trifallax are different species, and we propose that the oligotrich Halteria grandinella be reclassified as a hypotrich. This work also extends the available data base of eukaryotic DNA polymerase alpha sequences, and suggests new amino acid sequence targets for mutagenesis experiments to continue the functional dissection of DNA pol alpha biochemistry at the molecular level.