Following the earlier detection of six new esterified fumonisin B₁ (EFB₁) isomers containing three acyl groups in a Fusarium verticillioides-inoculated rice culture, it was assumed that linoleic, palmitic or oleic acid esterifies one of the free OH groups on the fumonisin backbone. On the basis of the results of our recent investigations we now propose that these EFB₁ isomers are actually 3-O- and 5-O-acyl derivatives of FB₁ (3-O-linoleoyl-FB₁, 5-O-linoleoyl-FB₁, 3-O-palmitoyl-FB₁, 5-O-palmitoyl-FB₁, 3-O-oleoyl-FB₁ and 5-O-oleoyl-FB₁). A F. verticillioides strain was identified that produced not only O-acyl-FB₁ isomers, but also low amounts of three N-acyl derivatives (N-linoleoyl-FB₁, N-palmitoyl-FB₁ and N-oleoyl-FB₁), which eluted from the HPLC column after the six O-acyl compounds and in the same sequence as for the O-acyl compounds. The characteristic positive and negative ESI-MS/MS spectra obtained after solid-phase extraction of the culture extract facilitated identification of these N-acyl-FB₁ derivatives. The biosynthesis of N-palmitoyl-FB₁ by F. verticillioides was verified by spiking the culture extract with synthetic N-palmitoyl-FB₁. This is the first report of the separation and mass spectrometric identification of the six O-acyl- and three N-acyl-FB₁ derivatives extracted from a F. verticillioides culture.