Ouabain has recently been identified in mammalian plasma with an apparent adrenocortical origin. The objectives of the present study were to determine whether boiled plasma supernatants (BPS) from spontaneously hypertensive rats (SHR) contained elevated levels of material able to inhibit 86Rb uptake, an indicator of sodium pump activity, compared with Wistar-Kyoto rats (WKY). Furthermore, the effect of increasing dietary calcium content from 1 to 3% on 86Rb-uptake inhibitory activity in plasma was examined. BPS from SHR and WKY consuming 1% calcium contained sodium pump inhibitory activity equivalent to 16.43 +/- 0.23 and 5.08 +/- 0.10 ng ouabain/ml, respectively (P < 0.0001). Increasing dietary calcium intake to 3% reduced plasma ouabain-like activity (OLA) to 9.97 +/- 0.20 ng/ml (P < 0.0001) in SHR but was without effect in WKY (5.39 +/- 0.05; not significant). It was then determined whether the plasma 86Rb-uptake inhibition could be attributed to authentic ouabain. In WKY plasma pools the percentage of OLA attributable to authentic ouabain was 38.0% by radioimmunoassay and 56.7% by 86Rb-uptake assay. In SHR these values were 3.8% and < 7.1%, respectively. Whereas the data in the present study provide confirmation of the presence of ouabain in rat plasma, ouabain does not account for the elevated OLA in SHR plasma reported here and elsewhere. This hypertensinogenic cardiotonic steroid appears to be appropriately downregulated in SHR rats.