Sequestration of transcription factors in the membrane is emerging as an important mechanism for the regulation of gene expression. A handful of membrane-spanning transcription factors has been previously identified whose access to the nucleus is regulated by proteolytic cleavage from the membrane. To investigate the existence of other transmembrane transcription factors, we analyzed computationally all proteins in SWISS-PROT/TrEMBL for the combined presence of a DNA-binding domain and a transmembrane segment. Using Pfam hidden Markov models and four transmembrane-prediction programs, we identified with high confidence 76 membrane-spanning transcription factors in SWISS-PROT/TrEMBL. Analysis of the distribution of two proteins predicted by our method, MTJ1 and DMRT2, confirmed their localization to intracellular membrane compartments. Furthermore, elimination of the predicted transmembrane segment led to nuclear localization for each of these proteins. Our analysis uncovered a wealth of predicted membrane-spanning transcription factors that are structurally and taxonomically diverse, 56 of which lack experimental annotation. Seventy-five of the proteins are modular in structure, suggesting that a single proteolysis may be sufficient to liberate a DNA-binding domain from the membrane. This study provides grounds for investigations into the stimuli and mechanisms that release this intriguing class of transcription factors from membranes.