Two different approaches have been utilized to synthesize cephaloglycin using immobilized-stabilized penicillin G acylase derivatives. These are thermodynamically and kinetically controlled strategies. The thermodynamically controlled strategy could be employed to synthesize cephalotin or penicillin G, but this approach in the synthesis of cephaloglycin presented serious difficulties because of the absence of conditions where the thermodynamics of the process and the enzyme activity/stability properties were good enough. The kinetically controlled strategy has given much better results. The systematic study of the different parameters that defined the maximum yields for this strategy has permitted the identification of its main problem as the hydrolysis of the antibiotic. Because of the rapid enzymatic hydrolysis of cephaloglycin that has been previously synthetized, the yields were poor and they decreased very rapidly after reaching the maximum yield. Three different strategies have been used to decrease this amidase activity (an excess of acyl donor, selection of acidic pH, and distortion of the enzyme molecule by methanol). Simultaneous utilization of these strategies has significantly improved this synthetic process with very high yields (around 95%), reaction rates, and enzyme stability.