Protein tyrosine kinases play a fundamental role in signal transduction pathways. Deregulated tyrosine kinase activity has been observed in many proliferative diseases (e.g., cancer, psoriasis, restenosis, etc.). Tyrosine kinases are, therefore, attractive targets for the design of new therapeutic agents against cancer. We have built up a pharmacophore model of the ATP-binding site of the epidermal growth factor receptor (EGFR) kinase and used it for the rational design of kinase inhibitors. Several examples of the successful use of this model are presented in this review. Amongst these, 4-substituted-pyrrolo[2,3-d]pyrimidines, a new class of highly potent and selective inhibitors of the EGFR kinase, have been identified and further optimized. The most active derivatives inhibited the EGFR tyrosine kinase with IC50 values between 1 and 5 nM. In EGF-dependent cellular systems, tyrosine phosphorylation, as well as c-fos mRNA expression, was inhibited with similar IC50 values. Further successful application of this pharmacophore model led to the identification and optimization of phenylamino-pyrazolo[4,3-d]pyrimidines and substituted isoflavones and quinolones, other classes of potent, selective, and ATP competitive EGFR kinase inhibitors with IC50 values in the low nanomolar range. Structure-activity relationships of both classes are discussed.