A series of 6-substituted 4-anilinopyrimido[5,4-d]pyrimidines has been prepared and shown to be potent inhibitors of the tyrosine kinase activity of the epidermal growth factor receptor (EGFR). These compounds are structurally related to the pyrido[3,2-d]- and pyrido[3,4-d]-pyrimidines previously shown to be EGFR inhibitors. Their structure-activity relationships (SAR) for inhibition of the isolated enzyme more closely resemble those of the [3,2-d] than the [3,4-d] pyridopyrimidine isomers. This suggests the requirement of an aza atom in the 7- but not the 5-position (i.e., a carbon atom in the 5-position) for the enhanced potency shown by 6-N-methylated derivatives in each series. X-ray crystal structures were determined for the three NHMe derivatives 2, 3, and 5c in the pyrido[9,2-d]-, pyrido[3,4-d]-, and pyrimido[5,4-d]-pyrimidine series, respectively. These show that a carbon rather than a nitrogen atom at the 5-position leads to significant conformational changes in the molecule (a longer C5a-C4 bond and a 30 degrees out-of-plane rotation of the phenyl group), due to the requirement to relieve nonbonding interactions between the C5 and N9 protons. Pyrimido[5,4-d]pyrimidine analogues bearing bulky, weakly basic solubilizing side chains linked to the 6-position through a secondary amine generally retained potency both against the isolated enzyme and for inhibition of autophosphorylation of EGFR in intact A431 cells. This agrees with a recent binding model that suggests this general class of compounds binds to EGFR with the 6-position located in an area of comparative bulk tolerance at the entrance to the ATP-binding pocket. While these solubilized pyrimido[5,4-d]pyrimidine analogues were less potent than the NHMe derivative 5c in the isolated enzyme assay, some were considerably superior to 5c (and among the most potent ever reported) as inhibitors of EGFR autophosphorylation in cellular assays.