Techniques are described in detail for the radioimmunological measurement of prostaglandin (PG) E2 and F2alpha in human urine. 50 ml urine samples are extracted with an organic solvent system and then purified by silicic acid column chromatography. The overall recovery after extraction and purification, calculated with labeled as well as unlabeled compounds, is in the order of 70%. The column eluates are assayed at 1:12-1:60 dilution in the "standard diluent" of the assay: 8 pg PGE2 or PGF2 alpha/ml of whole urine represents the lowest measurable concentration. A urine blank and a solvent blank were evaluated separately, subjecting 50 ml of urine obtained from an indomethacin treated subject ("PG-free" urine) or 50 ml of distilled water, respectively, to the extraction-purification procedures. Both were found not to interfere with the antigen-antibody reaction. Urinary PG-like immunoreactivity (LI) was characterized in terms of immunochemical and thin-layer chromatographic (TLC) behavior. Both urinary PGE2-LI and PGF2 alpha-LI behaved as authentic PGE2 and PGF2 alpha, upon dilution and on TLC. In 33 healthy female subjects (aged 19-58 yr), urinary excretion rates averaged 178 +/- 80 (mean +/- SD) ng/day for PGE2 and 498 +/- 181 ng/day for PGF2 alpha. In a group of 8 healthy men, both PGE2 and PGF2 alpha excretion rates were higher and more scattered than the female values. When two healthy women were given indomethacin (200 mg/day), urinary PGE2 and PGF2 alpha dropped to undetectable levels during the 4th day of drug therapy. Intravenous injection of furosemide (50 mg) in a female volunteer was followed by an immediate rise of urinary sodium, PGE2, PGF2 alpha and PGE2/PGF2 alpha ratio and plasma renin activity. In a 10 year old girl with Bartter's syndrome, urinary PG excretion rate was elevated with a 3 times higher than normal PGE2/PGF2 alpha ratio. Indomethacin therapy resulted in a prompt drop of PG excretion rate and of plasma renin activity. These results show that a combination of adequate purification steps and antisera with optimal characteristics provides a reliable method for measuring PGE2 and PGF2 alpha in urine, and therefore a valuable tool to further our knowledge of the physiology and physiopathology of the renal PG-system.