Laboratories evaluated whether an interference was causing a false-positive PSA for the Immulite 2000 immunoassay after a time course of increasing prostate-specific antigen (PSA) in a post-prostatectomy patient led to salvage therapy, which had no effect on the elevated PSA. Serial dilutions of PSA for the patient sample (6.1 ng/mL; post-prostatectomy reference range: <0.1 ng/mL [undetectable]) were linear (r > 0.99). However, the PSA measurement was reduced to 0.1 ng/mL after pretreatment of the sample with heterophilic antibody blocking reagent. PSA was undetectable (<0.1 ng/mL) when measured using two alternative immunoassays. These results were consistent with the presence of heterophilic antibody interference for the Immulite 2000 assay. In this case, heterophilic antibody interference with PSA measurement must have originated during the period of post-prostatectomy monitoring, and the apparent progressive increases in PSA may have been due solely to the progressive increase of this heterophilic antibody assay interference. In the absence of clinical correlation, positive PSA monitoring results should always be assessed for heterophilic antibody interference for at least one time point.