Abstract Hybrids have been obtained by protoplast fusion of penicillin-producing strains of Aspergillus nidulans with strains of A. rugulosus which do not produce the antibiotic at detectable levels. Induced haploidization of the hybrids allowed the recovery of stable segregants, which were screened for penicillin production. Approximately half of the segregants from each cross produced penicillin, indicating segregation of one or several linked genes which are essential for penicillin biosynthesis. These essential genes were found to be located on linkage-group VI of A. nidulans by observing segregation in multi-marked strains. The penicillin-producing segregants showed a wide range of titres. Some segregants, which had recombinant phenotypes, produced significantly more penicillin than the A. nidulans parent.