A method for measuring cortisol, corticosterone, testosterone, progesterone, and 17-OH progesterone simultaneously in 2 ml of plasma has been developed especially for episodic secretion studies in which many frequent assays are required. Steroids were extracted from plasma, separated into 4 fractions by paper chromatography, and individual steroids in each fraction were measured by radioimmunoassay. The individual steroid radioimmunoassays were demonstrated to be sensitive, specific, accurate, and precise.Base line 8 AM plasma steroid levels were measured in normal males and menstruating females both before and after ovulation. The values obtained were in good agreement with those previously reported by other investigators using different methods.In order to establish the normal diurnal variation in both sexes, plasma steroid levels were measured at 20-min intervals throughout the day in a normal man and a preovulatory woman. When plasma steroid levels were plotted against time frequent intermittent peaks that were consistent with episodic secretion were observed. As a result of this study progesterone, corticosterone, testosterone, and 17-OH progesterone have been added to the list of hormones that are secreted episodically. Corticosterone peaks were well synchronized with cortisol peaks, but those for progesterone, testosterone, and 17-OH progesterone were not. In the male 17-OH progesterone and testosterone peaks correlated well. It was concluded from these observations that corticosterone is secreted directly into the bloodstream by the adrenal under ACTH control, and that the other steroids are secreted from non-adrenal sources under other controls. The synchrony between plasma testosterone and 17-OH progesterone levels in the male suggests a common origin, presumably the testes.