Using toxin-catalyzed ADP-ribosylation and specific immunoblots we examined whether the mass of G-protein subunits, Gα5, Gαi (includes Gαi2, Gαi3, and Gα0) and Gαβ, in glomerular membranes was altered by dietary protein intake. ADP-ribosylation catalyzed by cholera toxin (CT) or pertussis toxin (PT) detected significant amounts of Gαs or Gαi in glomerular membranes from rats fed a low (6% casein) or a high (40% casein) protein diet. There was no significant difference in Gαs content between glomerular membranes from low or high protein-fed rats. However, the amounts of Gαi were significantly lower in glomerular membranes from rats fed a high protein diet when compared to glomerular membranes from rats fed a low protein diet. Two isoforms of immunoreactive Gαs, 45 and 52 kDa proteins, were detected in glomerular membranes. The predominant isoform of Gαs was a 52 kDa protein. As with ADP-ribosylation, immunoblots showed no significant difference in Gαs content between glomerular membranes obtained from the two diet groups of rats. Also, immunoreactive Gαi2, Gαi3 and Gβ were present in glomerular membranes. The mass O fGαi2 and Gαi3 was significantly lower in glomerular membranes of rats fed a high protein diet than in those of rats fed a low-protein diet. The decreased mass of total Gαi, that is Gαi2 and Gαi3, was comparable to that seen with PT-catalyzed ADP-ribosylation. By contrast, Gβ content was significantly greater in glomerular membranes of rats fed a high protein diet than in those of rats fed a low protein diet. Gα0 was not immunodetected in glomerular membranes. In addition, any G-protein subunit examined in the present study was not detectable in cytosolic extracts of glomerular membranes. A decrease in Gαi2 and Gαi3 and an increase in Gβ may contribute in part to the greater production of eicosanoids by glomeruli of rats fed a high protein diet.