Three DNA regions (TE1, TE2 and the intron) regulate the ubiquitous expression of the alpha 1-tubulin gene of Drosophila melanogaster. In this report, we identify two proteins that bind these DNA regions. One is the previously characterized GAGA transcription factor and the other is a newly identified 62 kDa polypeptide, TBF1 (TE1-binding factor 1). Purified GAGA factor binds three sites in TE2 and at least three in the intron. TBF1 was purified from embryos and binds to both TE1 and TE2. Together, the two proteins produce the same DNase I footprints in TE1 and TE2 as does a nuclear extract that transcribes the gene accurately. These footprints cover most of the TE1 and TE2 DNA. Moreover, one binding site for each protein coincides with a site that activates transcription in vitro. The characteristics of the GAGA factor and the genes it regulates suggest roles these two proteins are likely to play in regulating ubiquitous expression.