The precursor of aspartame N-benzyloxycarbonyl-L-aspartic acid-L-phenylalanine methyl ester (N-CBZ-L-ASP-L-PheOMe) was synthesized using thermolysin from eutectic mixtures of the substrates N-benzyloxycarbonyl-L-aspartic acid (N-CBZ-L-Asp) and L-phenylalanine methyl ester (L-PheOMe). Eutectic, mixtures were easily formed by mixing the substrates in the presence of water and/or organic solvents as adjuvants. Among the tested solvents the hydrophilic, solvents dimethyl sulfoxide (DMSO) and 2-methoxy ethyl acetate (MEA) were the best adjuvants for enzymic reactions. A maximum conversion yield of 73.8% was obtained for a combination of 6% DMSO and 6% MEA. In addition to benzyloxycarbonyl (CBZ), L-Asp derivatives with other protecting groups were used. Reactions with N-CBZ-L-Asp or N-tei-t-butyloxycarbonyl (BOC)-L-Asp formed eutectic mixtures at approximately 0 degreesC, whereas reactions with hydrophobic N-Ac-L-Asp and N-9-fluorenyhnethyloxycabronyl (Fmoc)-L-Asp formed mixtures at 25 and 40 degreesC, respectively. High conversion yields of 74 and 78% were achieved for N-CBZ-L-Asp and N-BOC-L-Asp but low yields of 56 and 35% were obtained for N-AC-L-Asp and N-Fmoc-L-Asp. Thermolysin was most effective with a yield of 72% compared to fungal protease, subtilsin, alpha -chymotrypsin, papain, and pepsin with yields of 50% or less.