A unique post-translational modification of tubulin has previously been described in which a tyrosine residue is reversibly added to the C terminus of the alpha-tubulin subunit. We have prepared peptide antibodies that specifically react (shown by competitive immunoassay and Western blots) with the tyrosinated (Tyr) and nontyrosinated (Glu) forms of alpha-tubulin. Immunofluorescence with these antibodies demonstrated that the distributions of Tyr and Glu tubulin in fixed cells were markedly different. Tyr tubulin was found throughout the interphase network of microtubules and in the metaphase spindle, whereas Glu tubulin was present in a limited subset of interphase microtubules and was absent from the astral fibers of the metaphase spindle. Double immunofluorescence showed that Glu and Tyr microtubules comprised distinct subsets of the total cellular microtubules. These results suggest that tyrosination is involved in the establishment of separate populations of microtubules that may functionally distinct.