We report the isolation and sequencing of genomic clones encompassing the entire alpha-tubulin II gene from the human malaria parasite Plasmodium falciparum. This gene is closely related to, but significant different from the alpha-tubulin I gene that we have described previously. These two genes represent the entire complement of alpha-tubulin sequences in this organism and are expressed in a stage-specific manner. The alpha-II gene is present as a single copy and encodes a tubulin molecule with a predicted length of 450 amino acid residues (49.7 kDa). Like the alpha-I gene, it contains two introns, which are in identical positions to those of alpha-I, but are about one-third smaller. The deduced alpha-II protein is very similar to alpha-tubulin I (94.2% amino acid identity), except for notable differences across residues 40-45. In addition, unlike the great majority of alpha-tubulin genes (including alpha-I), alpha-II does not encode a terminal tyrosine residue. Using pulsed field gel electrophoresis we demonstrate that the two alpha-tubulin genes, together with the single beta-tubulin gene, are unlinked, all residing on different chromosomes. We assign alpha-I to chromosome 9, alpha-II to chromosome 4 and beta-tubulin to chromosome 10.