Alterations in the structure of the DNA-binding protein specified by gene 32 of bacteriophage T4 have been detected using partial trypsin digestion as a conformational probe. Limited tryptic hydrolysis of the gene 32 protein removes a fragment ("B" region), of 21 amino acids from the NH2 terminus and a 6,200-dalton fragment ("A" region) from the COOH terminus. Poly(dT), poly(dC), and single-stranded DNA increase the rate of tryptic hydrolysis of the "A" region but decrease the rate of tryptic hydrolysis of the "B" region. Oligonucleotides, which are too short to permit cooperative binding of the gene 32 protein, do not alter the rate of tryptic hydrolysis of either the "A" or "B" regions. A model which accounts for these findings requires that the "B" region be involved in gene 32 protein:gene 32 protein interactions when the gene 32 protein: DNA complex is formed. As a consequence of the gene 32 protein:DNA interaction, the "A" region should be able to participate more effectively in vivo and in vitro with other proteins involved in T4 DNA metabolism.