Protein phosphorylation responses in intact enterocytes were examined by stimulating 32Pi-labeled T84 cell monolayers with histamine and resolving proteins by two-dimensional gel electrophoresis. Histamine increases 32P-incorporation into two acidic proteins of Mr 83,000 and of Mr 29,000, designated p83 and p29. Labeling of p83 and p29 is also increased in cells exposed to ionomycin, but not in cells exposed to vasoactive intestinal peptide under conditions resulting in cAMP-mediated secretion and cAMP-stimulated protein phosphorylation. When T84 cell fractions are incubated with [gamma-32P]ATP, labeling of p83 is stimulated by Ca++, but not by cAMP. Thus, histamine stimulates Ca++-mediated protein phosphorylation during the regulation of Cl- secretion.