We and others have reported an association between raf-1 protein serine/threonine kinase activity and transformation of mammalian cells. Because constitutive tyrosine phosphorylation of specific polypeptides is, in general, indicative of the transformed state of cells, we investigated the effect of activation of raf-1 on phosphotyrosine-containing proteins in a human head and neck squamous cell carcinoma-derived cell line, PCI-06A. raf-1 expression and activity were modulated in PCI-06A cells by means of stable DNA transfection of either the entire coding domain of the human c-raf-1 cDNA (in the sense or antisense orientation) or a fragment of c-raf-1 cDNA coding for the kinase domain of raf-1. Our data showed that constitutive activation of raf-1 correlated with morphological transformation, whereas the inhibition of raf-1 expression and activity had no detectable effect on cell morphology as compared with the untransfected cells. Immunoprecipitation of whole-cell lysates with anti-phosphotyrosine antibody followed by anti-phosphotyrosine immunoblotting revealed four phosphotyrosine-containing proteins of approximately 129, 120, 110, and 63 kDa (I-IV, respectively) in the antisense c-raf-1 cDNA-transfected cells showing relatively diminished raf-1 activity but not in the transfectants expressing activated raf. We concluded that tyrosine phosphorylation of I-IV proteins is abrogated in PCI-06A squamous carcinoma cells transformed with constitutively active raf-1 protein kinase.