We report an ultrasensitive time-resolved immunofluorometric assay (TRIFA) for prostate-specific antigen (PSA). The assay is an improvement of our previous report (Clin Chem 1993;39:2108-14) and includes the utilization of two monoclonal antibodies and a one-step incubation period, which greatly reduces analysis time. The new method demonstrates a superior lower analytical limit of detection (< or = 1 ng/L), a wide dynamic range, absence of a hook effect at 10(6) ng/L PSA, and equimolarity for free PSA and PSA-antichymotrypsin complex. Also, we have compared several aspects of our TRIFA with a commercially available third-generation assay (Immulite). An evaluation of breast tumor cytosol extracts from 315 patients shows PSA immunoreactivity > 15 ng/g of total protein in 28% and 23% by TRIFA and Immulite analysis, respectively. Both methods demonstrate a significant association between breast tumor PSA immunoreactivity and progesterone and estrogen receptor positivity (P <0.001). Analysis of serum samples obtained for monitoring of postradical prostatectomy patients reveals significant PSA changes at concentrations undetectable by conventional methods. The significance of these results as well as the potential applications of ultrasensitive PSA assays in breast and prostate cancers are discussed.