Abstract Objectives. Placental site trophoblastic tumor (PSTT) commonly presents with low and variable concentration of hCG immunoreactivity in serum which can be difficult to differentiate from early stage choriocarcinoma/gestational trophoblastic neoplasm (GTN) or quiescent gestational trophoblastic disease (quiescent GTD). Nontrophoblastic malignancies such as germ cell tumors or other tumors secreting low hCG must also be considered in the differential diagnosis. Because treatments for these conditions are different, a means of differentiating PSTT from other diagnoses is important. We investigate the usefulness of hCG-free β-subunit to make this discrimination. Methods. Data collected on cases referred to the USA hCG Reference Service for consultation served as a basis for this retrospective analysis. There were 13 cases with histology proven PSTT and 12 with nontrophoblastic malignancy. hCG-free β-subunit was measured by immunoassay and reported as a proportion of total hCG (hCG-free β-subunit(%)). hCG-free β-subunit(%) results were determined for all histologically proven cases of PSTT and for the nontrophoblastic malignancies. Comparisons of hCG-free β-subunit(%) were made and compared with those of the 82 choriocarcinoma/GTN cases and 69 quiescent GTD cases. The accuracy of hCG-free β-subunit(%) to discriminate these malignancies was analyzed by investigating the areas under receiver-operating characteristics curve ± standard error. Results. hCG-free β-subunit(%) was the predominant hCG form in cases of PSTT (mean ± standard deviation, 60 ± 19%) and nontrophoblastic malignancies (91 ± 11%), thus discriminating these diagnoses from choriocarcinoma/GTN (9.3 ± 9.2%) and from quiescent GTD (5.4 ± 7.8%). The cutoff of >35% free β-subunit is proposed. At this cutoff, 100% detection at 0% false-positive is achieved. The accuracy of hCG-free β-subunit(%) for this discrimination is 100 ± 0%. At a proposed cutoff of >80%, the free β-subunit test will also distinguish PSTT from nontrophoblastic malignancy, with 77% detection at 23% false-positive or an accuracy of 92 ± 3.2%. Conclusion. Measurement of the proportion hCG-free β-subunit(%) was found to be useful in the diagnosis of PSTT using proposed cutoff values of >35% and >80%. While this finding needs to be confirmed by larger studies, it would be reasonable to measure hCG-free β-subunit(%) whenever the diagnosis of PSTT is considered.