The family of mitogen activated protein (MAP) kinases appear to play a central role in relaying signals generated by receptor protein tyrosine kinases (RPTK) from the cell surface to the nucleus. We previously demonstrated that undifferentiated and mitotically active crypt cells have high levels of tyrosine phosphorylated proteins (DR Burgess, W Jiang, S Mamajiwalla and W Kinsey. 1989. J. Cell Biol., 109: 2139) possibly due to the activation of RPTKs and also have high pp60c-src protein tyrosine kinase activity (CA Cartwright, SN Mamajiwalla, SA Skolnik, W Eckhart and DR Burgess. 1993. Oncogene. 8: 1033) when compared to differentiated, non-mitotic villus cells. Since activation of RPTKs leading to cell proliferation or differentiation involves activation of the Ras-MAP kinase pathway, we chose to determine in this study if the activity of the MAP kinases were also regulated during differentiation of normal adult enterocytes. Our data show that although the 42 kD MAP kinase (p42mapk) was expressed in both crypt and villus cells, it was phosphorylated on tyrosine and active only in the crypt cells. Our data further suggest that p42mapk is inactivated during differentiation, possibly by a protein tyrosine phosphatase. Immunofluorescence studies revealed that p42mapk localized to the nuclei in both undifferentiated and differentiated enterocytes and colocalized with phosphotyrosine containing proteins at the region of the junctional complex. These results suggest that p42mapk and its regulators are tightly controlled during enterocyte differentiation in vivo and implicate p42mapk as a key regulatory molecule in the normal development of the adult intestinal epithelium.