Monoclonal antibodies to the modified nucleoside N-[9-(beta-D-ribofuranosyl)purin-6-ylcarbamoyl]-L-threonine (t6A) have been produced and characterized. These antibodies were utilized in a radioimmunoassay to quantitate the levels of this modified nucleoside in the urine of patients with benign breast disease, primary breast cancer, and metastatic breast cancer. Eighty-nine % (16 of 18) of patients with metastatic breast cancer excreted higher quantities of t6A than did patients with benign breast disease or primary breast cancer. This modified nucleoside represents a new, potential marker not previously included in similar studies using high-performance liquid chromatography. The immunoassay approach for quantitating this nucleoside has the advantage over high-performance liquid chromatography in that the former is more compatible with a clinical laboratory setting, and it does not require pretreatment of the urine or sophisticated analytical equipment. The elevation of t6A levels in metastatic breast cancer patients indicates that it may be a more sensitive marker than previously studied modified nucleosides. Furthermore, t6A might be particularly useful for monitoring transition to the metastatic stage in patients already diagnosed as having breast cancer.