For the enzymatic synthesis of the antibiotic cephalexin, an activated acyl donor is generally used as one of the substrates (kinetically controlled approach); however, the thermodynamically controlled approach might be of interest since there is no need for activation of the acyl donor and less waste is produced. If the synthesis reaction can be combined with an effective product removal step, the thermodynamic approach can be beneficial. The thermodynamically controlled synthesis of cephalexin was studied at various pH values, solvent concentrations, and temperatures. With direct synthesis in water, only small amounts of cephalexin were formed (0.1 mm from 20 mm starting material by the Xanthomonas citri enzyme). Addition of water-miscible organic solvent had a positive effect on synthesis (by the Escherichia coli enzyme); the equilibrium concentration of cephalexin, however, was at best increased by a factor of 2–3 (in methanol and triglyme). The equilibrium antibiotic concentrations reported in this study were notably lower than the values reported in the literature. These differences originate from the improved analytical methods that are available nowadays. Low product concentrations were also found for other side-chains with an amino group at the α-position. Side-chains without this group can be coupled and give acceptable product concentrations. For these antibiotics, a thermodynamically controlled process may be an alternative to kinetically controlled coupling.