The polymerase chain reaction (PCR) has been used to amplify a 0.52 kb segment of Giardia intestinalis DNA, using primers specific for nucleotide sequences conserved within two genes (tsp11 and tsa417) that encode homologous, cysteine-rich trophozoite surface proteins. Using products amplified from axenic isolates belonging to genetic groups I and II (defined on the basis of allozyme electrophoresis data), restriction endonuclease analysis revealed both tsp11-like and tsa417-like fragments within all samples. The study also identified among the amplification products of group II organisms an additional fragment, containing a novel PstI site, that is not detected in the reaction products of group I isolates. The recovery of three distinct PCR products from each group II isolate was verified by cloning the fragments into the plasmid vector pGEM-7. Fragments containing the new PstI site possess the ClaI site common to both tsp11 and tsa417-like fragments, but they lack the HindIII site which characterizes tsp11-like fragments and also lack the PstI and KpnI sites which characterize tsa417-like fragments. Spot-blot analyses using cloned fragments of all three types as probes showed strong homologous hybridization but weak heterologous hybridization, indicating that each type differs substantially in nucleotide sequence from the others. Because the samples of Giardia DNA used in the PCR were purified from cultures that had been established from single trophozoites, the data indicate that individual trophozoites belonging to genetic group II possess three homologous genes defined by these related fragments. The presence of a PstI site in the amplified segment of the newly-discovered third gene of group II organisms provides a simple diagnostic means of differentiating group I and II isolates.