I-TevII is the homing endonuclease encoded by the sunY intron of bacteriophage T4. The enzyme cleaves an intronless sunY gene near the exon I-exon II junction, thereby initiating intron homing into its cognate intronless allele. Specifically, I-TevII cleaves its DNA target 13 to 15 nucleotides (nt) downstream of the sunY intron insertion site, generating 2-nl 3'-OH extensions. Here, we present evidence that I-TevII makes predominantly minor groove contacts in two regions of its recognition sequence, as does I-TevI, the other homing endonuclease encoded by phage T4. Following cleavage, I-TevII was shown to remain bound to one of its DNA products, suggesting possible additional roles for the endonuclease in the mobility process. Interestingly, two distinct conformational changes were detected by gel analysis in the DNA substrate following binding by I-TevII, one occurring in the absence of Mg2+, the second being dependent on the presence of Mg2+. The Mg(2+)-induced distortion accompanies a nick in one strand, and may serve to bring the cleavage site on the other strand into proximity with the catalytic domain of the protein.