The natriuretic, hypotensive, and immunological properties of atrial extracts (AE) prepared from male and female, prepubertal, adult, and old rats were evaluated. Groups of animals were killed at ages 15, 25, 32, 39, 46, 56, and 290 days of age, and the atria were homogenized and extracted in 1.0 M acetic acid. Boiled, lyophilized extracts were stored and subsequently evaluated by bioassay and RIA. No sex differences (P > 0.05) were observed in the natriuretic, hypotensive, or immunoreactive properties of AE from rats of identical age. On the basis of bioassay determinations, no significant differences (P > 0.05) were observed in the natriuretic activity of AE prepared from rats of different ages. In contrast, AE-induced hypotension was significantly lower in bioassay rats receiving AE from 290-day-old rats than in rats infused with AE from 15-, 39-, or 56- day-old animals. Despite this reduced hypotensive effect, significant differences in RIA or bioassay parallelism were not observed between the tissue extracts and a synthetic atrial natriuretic factor (ANF) standard. Estimation of ANF content revealed that the quantity of ANF per heart as well as per microgram of DNA rose steadily with age. Two independent methods of ANF quantification (i.e. rat bioassay and RIA) yielded different estimates of atrial ANF content except at 15 days of age where the bioassay/RIA ratio was approximately 0.95. At 39, 56, and 290 days of age the ratios were 0.51, 0.44, and 0.53, respectively. A significant age-dependent decline in biological activity was noted when the hypotensive and natriuretic responses were normalized to an equivalent quantity of immunoreactive ANF. On the basis of these findings, we conclude that significant agedependent differences exist in atrial ANF content and, more importantly, in the biological and immunological activity of rat AE. Since the composition of AE is not well defined, analysis of biological activity solely on the basis of RIA determinations may be insufficient to adequately evaluate the physiological properties associated with these extracts. © 1987 by The Endocrine Society.