The goal of our investigations is to define the genetic control of microtubule-based processes in a higher plant. The available evidence suggests that we have achieved our first objective: the characterization of the complete α-tubulin and β-tubulin gene families of Arabidopsis. Four additional α-tubulin genes (TUA2, TUA4, TUA5, and TUA6) of Arabidopsis have been cloned and sequenced to complete the analysis of the gene structure for all six α-tubulin genes detectable on DNA gel blots of Arabidopsis genomic DNA hybridized with α-tubulin coding sequences. TUA1 and TUA3 were characterized earlier in our laboratory. Noncoding gene-specific hybridization probes have been constructed for all six α-tubulin genes and used in RNA gel blot analyses to demonstrate that all six genes are transcribed. The six genes encode four different α-tubulin isoforms; TUA2 and TUA4 encode a single isoform, as do TUA3 and TUA5. Two-dimensional protein gel immunoblot analyses have resolved at least four α-tubulin isoforms from plant tissues, suggesting that all of the predicted TUA gene products are synthesized in vivo.