Our knowledge of the enzymes and genes involved in the biosynthesis of beta-lactam antibiotics has increased notably in the last decade. The purification to homogeneity of some of these proteins as well as their biochemical characterization has allowed some of them to be used for synthesizing many different penicillins and cephalosporin-like products in vitro. In this report we describe the most important advances in this field, placing special emphasis on the enzymatic synthesis of hydrophobic penicillins. The use of purified acyl-CoA: 6-aminopenicillanic acid (6-APA) acyltransferase (AT) from Penicillium chrysogenum and several acyl-CoA ligases obtained from different microbial origins has led to the reproduction "in vitro" of the last step involved in in penicillin biosynthesis. By coupling these enzymatic systems (AT and acyl-CoA ligases) an impressive number of beta-lactam antibiotics has been obtained. Thus, most of the known natural penicillins, many of the semisynthetic variants and others, which until now can only be obtained chemically, have been synthesized enzymatically from their natural precursors. Furthermore, the use of heterologous proteins in coupled systems has opened a new and exciting field in beta-lactam antibiotic research, lending new perspectives to the traditional methodology followed by antibiotic fermentation industries.