Levels of erythropoietin have been consistently demonstrated in urine collections from healthy adult males using a modification of the polycythemic mouse assay. This method is stable and reproducible, and it provides assay animals with low background erythropoiesis. Six hour collections of urine can be concentrated without apparent loss of activity and the entire daily excretion can be used in the assay. Quantitation has been achieved by comparing the response to a concentrate of normal urine directly to a linear dose/response curve of erythropoietin standard B. Based on 6 hour urine collections, erythropoietin excretion in the subjects tested averaged 4.0 units of erythropoietin standard B (range 1.2-9.5). In addition to wide unexplained variations in results seen from day to day, the data indicate that a diurnal pattern of excretion of the hormone may exist.