The 10-deacetylbaccatin III:10beta-O-acetyltransferase isolated from Taxus cuspidata regiospecifically transfers short-chain alkanoyl groups from their corresponding CoA thioesters to the C10 hydroxyl of 10-deacetylbaccatin III. This 10-O-acetyltransferase along with five other Taxus acyltransferases on the paclitaxel (Taxol) biosynthetic pathway and one additional Taxus-derived acyltransferases of unknown function were screened for 4-O-acetyltransferase activity against 4-deacetylbaccatin III, 7-acetyl-, 13-acetyl-, and 7,13-diacetyl-4-deacetylbaccatin III. These 4-deacyl derivatives were semisynthesized from the natural product baccatin III via silyl protecting group manipulation, regioselective reductive ester cleavage with sodium bis(2-methoxyethoxy)aluminum hydride, and regioselective acetylation with acetic anhydride. Assays with the 4-deacetylated diterpene substrates and acetyl CoA revealed the taxane 10beta-O-acetyltransferase was able to catalyze the 4-O-acetylation of 4-deacetylbaccatin III to baccatin III and 13-acetyl-4-deacetylbacatin III to 13-acetylbaccatin III, although each was converted at lesser efficiency than with the natural substrate. In contrast, this enzyme was unable to acetylate 7-acetyl-4-deacetylbaccatin III and 7,13-diacetyl-4-deacetylbaccatin III substrates at C4, suggesting that the C7 hydroxyl of baccatin III must remain deacylated for enzyme function. The biocatalytic transfer of an acyl group to the tertiary hydroxyl on the oxetane moiety at C4 of the taxane ring demonstrates that the regiochemistry of the 10beta-acetyltransferase is mutable.