We have developed a procedure for simultaneous immunofluorometric assay (IFMA) of prostate-specific antigen (PSA) and its complex with alpha 1-antichymotrypsin (ACT). A PSA-specific monoclonal antibody, which captures both free PSA and the PSA-ACT complex, was used as solid-phase antibody. Total PSA immunoreactivity was measured with a Eu(3+)-labeled PSA antibody that reacted with both free PSA and PSA-ACT. PSA-ACT was assayed simultaneously with a Sm(3+)-labeled polyclonal ACT antibody as a tracer. As standard we used pooled serum in which most of the PSA occurred as the PSA-ACT complex. The assay range was 0.03-500 micrograms/L for total PSA and 0.16-450 micrograms/L for PSA-ACT. In comparison with the assay for total PSA, assay of the PSA-ACT/PSA ratio improved the clinical specificity for cancer by reducing the number of false-positive results in prostatic hyperplasia.