Three subunits (srfAORF1, srfAORF2 and srfAORF3) of the Bacillus subtilis surfactin synthetase multi-enzyme complex have been identified by SDS-PAGE and Western blot analyses. In accordance with the sequence analysis of the surfactin (srfA) operon, the protein subunits have a molecular mass of 402,000 Da, 401,000 Da and 144,000 Da, respectively. Confirmation of the identity of the proteins was obtained by analysing the total protein content of a number of mutant strains which harbour deletions or insertions either in the srfA promoter or in different positions within the srfA operon. The three subunits were partially purified by means of a series of chromatographic steps including ion-exchange chromatography, hydrophobic chromatography and gel filtration chromatography. The partially purified proteins were used in activity assays to establish their amino-acid recognition specificity. In agreement with previously published results, this analysis showed that srfAORF1 recognizes glutamic acid and Leu, srfAORF2 recognizes Val, aspartic acid and Leu and srfAORF3 recognizes Leu. In addition, the subunits can activate and bind other amino acids, although with lower specificity. In particular, srfAORF1 binds Val, Ile and aspartic acid, srfAORF2 glutamic acid and Ile and srfAORF3 Ile and Val. Competition experiments as well as sequence comparison strongly suggest that the Leu binding sites of the three subunits can accept, beside Leu, Ile and Val. The kinetic parameters of srfAORF3 for Leu, Ile and Val have been determined.