Oligodeoxynucleotides have been prepared that contain changes in the functional group pattern present in the EcoRI recognition site. These changes involve "functional group deletions", "functional group reversals", and "displaced functional groups". Steady-state kinetic parameters have been used to characterize the interaction of these modified recognition sites with the EcoRI endonuclease. Changes in the functional group pattern have varying effects upon the cleavage reaction. Both the exocyclic amino groups of the two adenine residues and the methyl groups of the thymine residues appear to interact with the endonuclease quite differently. In both cases efficient catalysis was observed when these functional groups were present at the "outer" dA-dT base pair. Selectivity was decreased by over an order of magnitude largely via increases in Km when these functional groups were deleted. Similar modifications at the "inner" dA-dT base pair did not alter the kinetic parameters significantly from those observed with the native sequence. Addition of an amino group to the minor groove at the outer dA-dT base pair resulted in a modified recognition site that interacted with the enzyme, on the basis of observed competitive inhibition kinetics, but was not cleaved.