An assay for alpha-tubulin acetyltransferase (TAT) activity based on affinity isolation of labeled acetylated alpha-tubulin was developed for use with crude subcellular fractions. Microtubules were polymerized and immobilized on an anti-alpha-tubulin-agarose and then incubated with the subcellular fraction and [3H]acetyl-coenzyme A (CoA). The labeled product was eluted from the antibody-agarose and the tritiated acetate incorporation determined. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot of the eluate verified the presence of acetyl-alpha-tubulin. Analysis of bovine retinal fractions showed the highest specific activity of tubulin acetyltransferase activity in the 27,000g pellet fraction (P2) of retinal homogenates. This transferase activity was proportional to the concentration of microtubule protein immobilized under polymerizing conditions and had an apparent Km of 3 microM for acetyl-CoA. The activity was solubilized from the P2 pellet by a high ionic strength buffer. The properties of the retinal TAT determined by this assay are very similar to those reported for a more purified enzyme preparation from Chlamydomonas flagella using the conventional trichloroacetic acid precipitation assay and support the use of this method for samples in which high backgrounds prohibit use of the conventional assay.