NHE3 activity is regulated by phosphorylation/dephosphorylation processes and membrane recycling in intact cells. However, the Na(+)/H(+) exchanger (NHE) can also be regulated by G proteins independent of cytoplasmic second messengers, but the G protein subunits involved in this regulation are not known. Therefore, we studied G protein subunit regulation of NHE3 activity in renal brush-border membrane vesicles (BBMV) in a system devoid of cytoplasmic components and second messengers. Basal NHE3 activity was not regulated by G(s)alpha or G(i)alpha, because antibodies to these G proteins by themselves were without effect. The inhibitory effect of D(1)-like agonists on NHE3 activity was mediated, in part, by G(s)alpha, because it was partially reversed by anti-G(s)alpha antibodies. Moreover, the amount of G(s)alpha that coimmunoprecipitated with NHE3 was increased by fenoldopam in both brush-border membranes and renal proximal tubule cells. Furthermore, guanosine 5'-O-(3-thiotriphosphate) but not guanosine 5'-O-(2-thiodiphosphate), the inactive analog of GDP, increased the amount of G(s)alpha that coimmunoprecipitated with NHE3. The alpha(2)-adrenergic agonist, UK-14304 or pertussis toxin (PTX) alone had no effect on NHE3 activity, but UK-14304 and PTX treatment attenuated the D(1)-like receptor-mediated NHE3 inhibition. The ability of UK-14304 to attenuate the D(1)-like agonist effect was not due to G(i)alpha, because the attenuation was not blocked by anti-G(i)alpha antibodies or by PTX. Anti-Gbeta(common) antibodies, by themselves, slightly inhibited NHE3 activity but had little effect on D(1)-like receptor-mediated NHE3 inhibition. However, anti-Gbeta(common) antibodies reversed the effects of UK-14304 and PTX on D(1)-like agonist-mediated NHE3 inhibition. These studies provide concrete evidence of a direct regulatory role for G(s)alpha, independent of second messengers, in the D(1)-like-mediated inhibition of NHE3 activity in rat renal BBMV. In addition, beta/gamma dimers of heterotrimeric G proteins appear to have a stimulatory effect on NHE3 activity in BBMV.