Two methods of selecting penicillin overproducers were developed, based on the hypothesis that Penicillium chrysogenum mutants resistant to high concentrations of phenylacetic acid (PAA) could use this precursor more efficiently (and thus produce more penicillin). It was determined that 0.2% PAA was a selective concentration for pregerminated spores of P. chrysogenum P2. An alternative screening method, using young mycelia already in the production phase, was also developed. Applying the former method to a nitrosoguanidine mutagenized population, 12 resistant mutants were obtained, of which 2 (16.7% of the resistant population) produced more antibiotic than the parental strain. Using the second method, 14 resistant mycelial mutants were obtained, of which 7 (50% of the population) produced more than the original. Acyltransferase and cyclase activity were measured in the best strain obtained by each screening method. The results showed a twofold increase of cyclase activity in the mutant strains as compared with the original P2 strain, and a six-fold increase in acyltransferase activity in the mutant obtained by the mycelial screening method. It is concluded that both methods can be used for rational screening for penicillin-overproducing mutants.