The phosphorylation of rat renal brush border membrane protein was analyzed after incubation of cortical slices with 32P-orthophosphate and compared with the phosphorylation by γ-32P-ATP of isolated brush border vesicles. Phosphate incorporation into brush border membranes isolated from slices was linearly related to the incubation time as well as to the specific activity of orthophosphate present during slice incubation. Incorporation of phosphate into proteins reached an equilibrium after about 60min, whereas incorporation of phosphate into lipids increased continuously. In brush border membranes isolated from slices incubated with orthophosphate (32P), the addition of cAMP or theophylline produced a dephosphorylation of a 47,000-dalton protein; no increased phosphorylation was observed. In brush border membranes, phosphorylated with γ-32P-ATP, cAMP and dibutyryl cAMP (dB-cAMP) produced an increase in phosphorylation but no dephosphorylation. Sodium-dependent phosphate transport in brush border membranes was not altered by an incubation of slices with cAMP or dB-cAMP. These observations suggest that the phosphorylation machinery of isolated rat renal brush border membranes does not correspond with the mechanisms leading to phosphate incorporation into brush border membrane proteins in the intact cel