There is a growing body of evidence indicating that prostate-specific antigen (PSA) may be present in many steroid hormone-stimulated epithelial tissues other than that of the prostate. In particular, breast tumor cell lines treated with steroid hormone receptor agonists, breast tumors, and normal human breast have recently been found by our group to contain PSA. To investigate whether PSA may also be present in other human tumors, we employed a highly sensitive immunofluorometric assay technique to quantify PSA immunoreactivity in tumor extracts. Using a PSA-positivity cutoff value of 0.005 ng per mg of protein, 23 of 43 diverse tumors tested positive for PSA protein. Confirmatory analyses for PSA by a commercially available method (IMx) on six samples demonstrated a high degree of concordance between the two methods. To establish the molecular weight of the immunoreactive species, the most highly positive tumor extracts of each tumor type were fractionated by high performance liquid chromatography. Whereas the majority of tumors had immunoreactivity eluting at both 100 KDa and 33 KDa, corresponding to PSA bound to alpha 1-antichymotrypsin and free PSA, respectively, the colon and parotid tumors displayed immunoreactivity only at the 33 KDa fraction. We conclude that in addition to breast tumors and normal breast, colon, ovarian, liver, kidney, adrenal, and parotid tumors can also produce PSA. The physiological role of PSA in these tumors is currently under investigation.