The endogenous tyrosine protein kinase activity (TPKA) associated with brush-border (BBM) and basolateral (BLM) membranes of rat kidney cortex was studied with an anti-phosphotyrosine monoclonal antibody (PY20). Distinct major phosphotyrosine-containing proteins were associated with BBM (50, 54, and 120 kDa) and BLM (37, 90, 130, and 170 kDa). For both plasma membranes, tyrosine phosphorylation leveled off after 10 min of incubation. Endogenous phosphotyrosine-specific protein phosphatases (PT-Pases) were active in both membranes, since the presence of sodium vanadate or ammonium molybdate, which are inhibitors of PTPases, was essential to detect endogenous phosphorylation. Substrates and/or tyrosine protein kinases (TPKs) seem to be differently distributed in these plasma membranes, since phosphorylation of endogenous substrates in BLM and BBM was differently sensitive to competitive inhibitors of TPKs. Moreover, insulin- and insulin-like growth factor I-stimulated tyrosine phosphorylation of a 90-kDa substrate was only observed in solubilized BLM proteins. However, similar p60v-src-related TPKs appear to be present in the BBM and BLM, since an antibody raised against p60v-src recognized proteins of 52, 58, and 75 kDa by immunoblotting and could immunoprecipitate the TPKs associated with both plasma membranes. These data provide evidence that the endogenous tyrosine protein phosphorylation observed in the BLM is catalyzed by nonreceptor TPKs as well as receptor TPKs, whereas that observed in the BBM is exclusively due to nonreceptor TPKs.