Data on the involvement of aldosterone in the regulation of the renin-angiotensin-aldosterone system (RAAS) in rodents are still scarce, partly due to the high sample volumes needed by commercially available assays and to the very low aldosterone concentrations present. We have developed a highly sensitive and non-isotopic immunoassay, requiring a volume of only 50 microl serum for a duplicate measurement, employing a highly specific monoclonal antibody against aldosterone. The assay was validated in human and mouse samples and exhibited a linear working range from 10 to 1000 pg/ml. Values obtained after a chromatographic purification step correlated significantly to the dichloromethane extraction ordinarily used. Basal aldosterone values were measured in 75 mouse hybrids and found within the linear range (173+/-21 pg/ml), with no significant difference between males and females. Additionally, we show an increase in serum aldosterone in mice from 3 to 11 weeks of age. Mice of the same genetic background were treated with dexamethasone intraperitoneally (n=7), resulting in significantly decreased concentrations (35+/-3 vs 114+/-33 pg/ml in controls; P<0.001). In contrast, adrenocorticotropic hormone resulted in significantly increased serum aldosterone (603+/-119 pg/ml; n=7; P<0.001), as did the physiological stimulation of the RAAS by a high K(+)/low Na(+) diet (1369+/-703 vs 172+/-36 pg/ml). In conclusion, we have developed and validated an extremely sensitive assay for determination of aldosterone concentrations from very small serum samples, which could be especially useful in pharmacological intervention studies in rodent models.