Naturally-occurring hydroxybutinylbithiophene derivatives were acylated by enzyme preparations of Tagetes patula seedlings in the presence of distinct acyl-Coenzyme A esters. The O-acyltransferase activity could only be detected after almost complete separation of the enzyme from counter-currently acting esterases which were present in the same extracts. This was achieved by affinity chromatography on Cibachron Blue A. During this procedure, the O-acyl-transferase was split, yielding two active fractions. Both had a Mr of 37,000 (±5,000), equal isoelectric properties, a pH optimum of pH 7.0, and were considerably inhibited in the presence of free Coenzyme A. Small differences existed in their affinities for their thiophenic substrates (3,4-dihydroxybutinylbithiophene and 4-hydroxybutinylbithiophene, respectively), as well as for various acyl-CoA esters as cosubstrates. With the preferred cosubstrate, acetyl-CoA, acylation took place at the 4-position of the butinyl side chain of the molecules, forming the naturally- occurring 4-acetoxybutinylbithiophene and 3-OH,4-OAc-butinylbithiophene, respectively. From the other acyl-CoA esters employed, only propionyl-CoA was likewise converted, forming the corresponding O-propionyl esters. The reactions observed are suggested to be catalyzed by two acetyl-CoA: 4-hydroxybutinylbithiophene O-acetyltransferase isoenzymes which exhibit different affinities for particular substrates and cosubstrates. The activities of both the isoenzymes changed drastically if plant material from different developmental stages was used as enzyme source. Therefore, it may be suggested that these isoenzymes play an important regulatory role in the metabolism of naturally-occurring hydroxy- and acetoxybutinylbithiophenes and their derivatives.