Cell-free extracts of Streptomyces clavuligerus contain the enzyme deacetoxycephalosporin C synthase ("expandase"), which catalyzes the oxidative ring expansion of the natural substrate delta-o-alpha-aminoadipyl-6-APA (penicillin N) into the primary cephalosporin, deacetoxycephalosporin C in the presence of magnesium ions, ferrous ions, ascorbate, and a-ketoglutarate. Eighteen unnatural side chain analogues of penicillin N were exposed to these reaction conditions. Only D-carboxymethylcysteinyl-6-APA was found to undergo ring expansion. Of special interest is the observation that adipyl-6-APA and m-carboxyphenylacetyl-6-APA were not expanded.