In order to understand the functional significance of the transmembrane domain of TrwB, an integral membrane protein involved in bacterial conjugation, the protein was purified in the native, and also as a truncated soluble form (TrwBDeltaN70). The intact protein (TrwB) binds preferentially purine over pyrimidine nucleotides, NTPs over NDPs, and ribo- over deoxyribonucleotides. In contrast, TrwBDeltaN70 binds uniformly all tested nucleotides. The transmembrane domain has the general effect of making the nucleotide binding site(s) less accessible, but more selective. This is in contrast to other membrane proteins in which most of the protein mass, including the catalytic domain, is outside the membrane, but whose activity is not modified by the presence or absence of the transmembrane segment.