Unilateral ureteral obstruction markedly alters sodium and water reabsorption by the affected kidney. These abnormalities may be due, at least in part, to decreased Na,K-ATPase activity in various segments of the nephron during obstruction. The reason for this decreased activity has, however, remained speculative. The present study examines the potential mechanisms underlying the decreased Na,K-ATPase activity in obstruction. The Na,K-ATPase activity was markedly reduced in basolateral membrane vesicles prepared from the cortex of the obstructed kidney of rats with unilateral ureteric obstruction of 24 hours duration when compared to basolateral membrane vesicles from contralateral kidneys of the same rats or to basolateral membrane vesicles from kidneys of sham operated animals. However, no such difference was present three days post-release of unilateral ureteric obstruction. When basolateral membrane vesicles were incubated with sodium dodecyl sulphate to permeabilize the vesicles, no difference in the proportion of enzyme latency was detected between the basolateral membrane vesicles from obstructed kidneys and those from sham operated rats. Immunoblotting with antibodies to the alpha subunit of Na,K-ATPase revealed equal amounts of enzyme in the basolateral membrane vesicles from contralateral kidneys, obstructed kidneys and sham operated rats. When incubated with liposomes under conditions conducive to fusion and lipid exchange the activity of Na,K-ATPase in basolateral membrane vesicles from obstructed kidneys was reconstituted almost to normal levels. This increase in enzyme activity did not occur in basolateral membranes from contralateral kidneys or in membranes from kidneys of sham operated rats incubated in the same manner. We conclude that the reduction in Na,K-ATPase activity in ureteric obstruction is not related to reduced quantity of enzyme or to its sequestration in impermeable vesicles, but to changes in the lipid environment of the basolateral membrane.