During late pregnancy, the rat undergoes massive plasma volume expansion due to cumulative renal sodium retention. In the present study, conducted in virgin, mid- (days 11-13), and late-pregnant (days 18-20) rats, we measured both Na+-K+-ATPase activity (by coupled enzyme assay) and abundance of the alpha-subunits of the Na+-K+-ATPase (by Western and slot blot analyses) in renal cortex, medulla, and brain stem. Unexpectedly, Na+-K+-ATPase in renal cortex, in both stages of pregnancy, is reduced versus the virgin, consistent with our finding of a reduced quantity of the alpha1-subunit. In renal medulla, there is a small rise in activity at midterm, but there is no difference in either activity or abundance of the alpha1-subunit in late pregnancy, when renal Na retention is maximal. In brain stem, where only alpha2- and alpha3-subunits are evident, pregnancy has no impact on enzyme activity or abundance of either isoform. In conclusion, the outcome of these experiments was unexpected in that we did not observe increased renal Na+-K+-ATPase activity in late pregnancy in the rat. In fact, in renal cortex, Na+-K+-ATPase activity and abundance are reduced. Whatever promotes net sodium retention in pregnancy must be capable of overwhelming this and several other strong natriuretic signals.