The alpha-tubulin cDNA clone, pTUB5 (1536 bp), contains the entire coding sequence for an alpha-tubulin of 450 amino acids (mol. wt. 50,000) and is the first tubulin sequence to be reported for a fish. The transcript encoded by pTUB5 showed testis-specific expression. Its gene appears to be present at a single copy number in the trout tubulin multigene family. Comparison of the TUB5 amino acid sequence to the murine testis-specific alpha-tubulin (M alpha 3/7) revealed that eight of ten murine isotype-specific amino acid substitutions were in common. Certain of these substitutions are also conserved in other testis-specific alpha-tubulins and in alpha-tubulins of certain protozoans with flagella. The conservation of specific amino acids at these positions suggests that they may have important structural and or functional roles in the microtubules of axonemes.