We have demonstrated that phorbol esters such as phorbol dibutyrate (PhE) transiently inhibit Na/H exchange both in intact avian enterocytes and in brush border membrane (BBM) vesicles prepared from enterocytes treated with PhE (Chang et al., 1991, Am. J. Physiol. 260: C1264-C1272). Maximal inhibition occurs at 90 sec and values return to baseline by 15 min. In this study we examined if PhE causes changes in BBM protein phosphorylation by two methods: 1) in situ phosphorylation in which intact cells prelabeled with ''PI were treated with PhE; 2) in vitro phosphorylation in which BBM, isolated from untreated and PhE-treated enterocytes, were exposed to v~~P-ATP. In situ phosphorylation studies showed that, at 90 sec, PhE increases the phosphorylation of BBM proteins of M, (pl): 150 (6.5), 89 (-6.2), and 48 (=6.1) kDa which declined to control values at 15 min, suggesting that these may be transport-related substrates. These labeled substrates were recovered in the detergent-insoluble fraction after extraction with 0.1 % Triton X-100 overnight. Transient phosphorylation of a number of proteins was also observed when B B M prepared from control or PhE-treated cells were incubated with y 32P-ATP 2 10 nM PhE, phosphatidyl serine, Ca2+, and/or exogenous