Nuclear protein extracts from Mu-active, Mu-inactive and non-Mutator lines of maize were used to identify the binding sites for maize nuclear proteins in the terminal inverted repeats (TIR) of the Mu1 transposable element. We found binding activities of nuclear proteins that specifically interact with both TIRs of the Mu1 element. DNase I footprinting was performed to localize the binding sites. We found that the nuclear proteins from Mu-active lines and non-Mu lines bound to the Mu1 TIR at two different sites, i.e. a 13 bp sequence (CGGGAACGGTAAA, designated as site I) and another 8 bp sequence (CGGCGTCT, designated as site II). However, the nuclear proteins from Mu-inactive lines bound only one of these sites, i.e. site I. Mobility shift assays with synthetic oligonucleotides containing site I and II respectively confirmed the specificities of these binding activities. Site I was shown to be an imperfect direct repeat of a hexamer binding site (CGGGAACGGTAA). Oligonucleotides containing either of the hexamers showed specific binding activity to nuclear protein from both Mu-active and Mu-inactive lines. The possible role of these proteins in Mu transposition is discussed.