We are developing and refining germline transformation systems in the red flour beetle, Tribolium castaneum (Herbst), based on the piggyBac transposable element. As one component of this effort, we are evaluating various native Tribolium promoters, including that of alpha tubulin, for driving expression of piggyBac transposase. We have cloned and sequenced a Tribolium castaneum alpha tubulin gene. After examination of a 741-bp segment immediately upstream of the transcription unit, a putative promoter was identified. Like Drosophila alpha tubulin promoters, this putative Tribolium promoter has consensus CAAT and TATA boxes just upstream of the predicted transcriptional start site. In addition, the Neural Network Promoter Prediction program ("http://www.fruitfly.org/seq_tools/promoter.html") gave this sequence a score of 0.92, indicating a high likelihood that the promoter motifs are authentic. Finally, we have numerous Tribolium alpha tubulin cDNAs that begin within 20 bases of the predicted site. This alpha tubulin promoter was attached upstream of a reporter gene, vermilion (tryptophan oxygenase), which rescues eye pigment in a vermilion mutant (pigment-deficient) strain (Lorenzen et al., 2002, Insect mol Biol 11: 399-407). Alpha tubulin promoter elements capable of driving vermilion in transient assays will be used to drive green fluorescent protein (to determine spatial and temporal expression patterns), and piggyBac transposase (to remobilize piggyBac donor elements).