Na-K- and Mg-activated ATPase activities were determined in maculae densae and glomeruli dissected from both superficial and juxtamedullary nephrons of normal rabbits, using an ultramicro method including a cycling reaction. Activities were expressed as Pi generated per macula densa or per glomerulus and normalized for tissue volume. Results indicate that the mean volume of superficial and juxtamedullary macula densa samples was not statistically different, while glomeruli from deep nephrons had sample volumes that were 29% larger than those from superficial nephrons (P less than 0.001). Correcting for volume both superficial and juxtamedullary macula densa samples had an Na-K-ATPase activity of 0.37 +/- 0.21 fmol X h-1 X (micron3)-1 X Mg-ATPase activity in both pools was also similar [0.41 +/- 0.07 and 0.52 +/- 0.1 fmol X h-1 X (micron3)-1]. Na-K-ATPase activity in macula densa cells is estimated to be about 1/40th the activity of surrounding cortical thick ascending limb cells. Total glomerular ATPase per unit volume was significantly higher in glomeruli from superficial than from deep nephrons [0.41 +/- 0.04 vs. 0.28 +/- 0.04 fmol X h-1 X (micron3)-1, P less than 0.05]. There was no statistically significant activity of Na-K-ATPase in either superficial or deep glomeruli. These results suggest that in contrast to previous reports, the macula densa contains Na-K-ATPase, but at a low level relative to surrounding tubular cells. Further, in normal rabbits, this activity is invariant in superficial and juxtamedullary samples.